Compositions for inhibiting redox-sensitive GTPases

ABSTRACT

The invention provides compositions for inhibiting a redox-sensitive GTPase protein, including a Rho or Rab family GTPase, comprising an effective amount of a redox-sensitive purine compound and an effective amount of a redox agent. The invention further provides methods of inhibiting a redox-sensitive GTPase protein, including a Rho or Rab family GTPase, by administering compositions of the invention. Methods of screening for compounds that inhibit a redox-sensitive GTPase protein, including compounds that target and inhibit Rho or Rab family GTPases, are further provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Appl. No.61/426,643, filed Dec. 23, 2010. The content of the aforesaidapplication is relied upon and incorporated by reference in itsentirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the sequence listing (Name: SEQ LISTING ST25.txt, Size:5,828 bytes; and Date of Creation: Mar. 19, 2012) electronicallysubmitted via EFS-Web is incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The field of the invention relates to therapeutics. In particular, thefield of the invention relates to the use of a redox-sensitive purinecompound in combination with a redox agent to inhibit redox-sensitiveGTPases, including Rho or Rab family GTPases.

2. Description of the Related Art

Small GTPases, including the Rho and Rab family GTPases, are involved invarious cellular signaling events. Rho and Rab family GTPases belong tothe Ras superfamily of GTPases. Rho and Rab GTPases, like all members ofthe Ras superfamily, function by cycling between inactive GDP-bound andactive GTP-bound states (FIG. 1). Heo, J. (2011) Antioxid. RedoxSignaling 14, 689-724. Various protein regulators such as guaninenucleotide exchange factors (GEFs) and GTPase-activating proteinscontrol this GDP/GTP cycling. GEFs enhance the guanine nucleotideexchange (GNE) of these GTPases. Bos et al. (2007) Cell 129, 865-877.Dbl's big sister (Dbs), one of the Rho-specific GEFs, has been shown tobe a RhoC GEF. Dietrich et al. (2009) Biol. Chem. 390, 1063-1077.Because cells contain relatively higher concentrations of GTP than GDP(Traut, T. W. (1994) Mol. Cell. Biochem. 140, 1-22), the GEF-mediatedGNE of these GTPases populates the GTP-loaded active GTPases in cells.GTPase-activating proteins stimulate hydrolysis of the γ-phosphate ofthe bound GTP to produce inactive GDP-bound GTPases and free phosphates.Bos et al. (2007) Cell 129, 865-877.

Several Rho family proteins have been identified thus far, includingRhoA, RhoB, RhoC, RhoG, Rac1, Rac2, Rac3, Cdc42, TC¹⁰, TCL. Rho familymembers modulate various cellular processes, including cell morphology,movement and proliferation by mediating distinct cytoskeletal changes.Etienne-Manneville et al. (2002) Nature 420, 629-635. Misregulation ofRho GTPases has been implicated in many disorders, including cancer,heart and lung diseases, vascular diseases and diseases of the immunesystem. See e.g., van Leeuwen et al. (1995) Oncogene 11, 2215-2221;Fritz et al. (1999) Int. J. Cancer 81, 682-687; Boettner et al. (2002)Gene 286, 155-174; Benitah et al. (2003) Rev. Oncol. 5, 70-78; Numaguchiet al. (1999) Circ. Res. 85, 5-11; Laufs et al. (2000) Circ. Res. 87,526-528; Satoh et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103,7432-7437; Faried et al. (2006) Eur. J. Cancer. 42, 1455-1465; Touge etal. (2007) Int. J. Oncol. 30, 709-715.

RhoA has increasingly attracted clinical interest because of theemerging evidence of its role in the pathogenesis of several bloodvessel diseases, including hypertension and atherosclerosis.Rabinovitch, M. (1991) Toxicol. Pathol. 19, 458-469; Numaguchi, K.(1999) Circ. Res. 85, 5-11; Laufs et al. (2000) Circ. Res. 87, 526-528;Alvarez de Sotomayor et al. (2001) Eur. J. Pharmacol. 415, 217-224;Kuzuya et al. (2004) J. Cardiovasc. Pharmacol. 43, 808-814; Kontaridiset al. (2008) Circulation 117, 1423-1435. Accordingly, it is consideredan important target for future therapeutic agents. Budzyn et al. (2006)Trends Pharmacol. Sci. 27, 97-104; Shimokawa et al. (2007) TrendsPharmacol. Sci. 28, 296-302. Activation of RhoA results indownregulation of the myosin light chain phosphatase (MLCP) viaupregulation of Rho kinase (ROCK). Halka et al. (2008) Cardiovasc.Pathol. 17, 98-102; Barman et al. (2009) Vasc. Health Risk Manag. 5,663-671. Inactivation of the MLCP in turn populates its downstream MLCtarget in its dephosphorylated state, resulting in induction ofvasorelaxation.

RhoC has increasingly attracted clinical interest because of theemerging evidence of its metastatic role in inflammatory breast cancer(IBC), which is the most lethal form of locally advanced breast cancerand annually accounts for approximately 6% of the new breast cancercases in the United States. Jaiyesimi et al. (1992) J. Clin. Oncol. 10,1014-1024. RhoC has been shown to be overexpressed in ˜90% of human IBClesions. van Golen et al. (1999) Clin. Cancer. Res. 5, 2511-2519; Clark,E. A., et al. (2000) Nature 406, 532-535. RhoC may also play a role inmetastasis of various other tumors including hepatocellular and coloncarcinomas. Wang et al. (2003) World J. Gastroenterol. 9, 1950-1953;Bellovin et al. (2006) Oncogene 25, 6959-6967.

Rab GTPases function via their specific effectors (Stenmark et al.(2001) Genome. Biol. 2, reviews 3007.3001-.3007; Jordens et al. (2005)Traffic 6, 1070-1077; Sandilands et al. (2008) Trends Cell Biol. 18,322-329; Stenmark, H. (2009) Nat. Rev. Mol. Cell Biol. 10, 513-525) asregulators of distinct steps in membrane traffic pathways, includingregulation of vesicle formation and movement. As with various RasGTPases, misregulation of Rab GTPases results in development of avariety of cancers. Stein et al. (2003) Adv. Drug Deliv. Rev. 55,1421-1437; Chia et al. (2009) Biochim. Biophys. Acta 1795, 110-116.

When small GTPases are redox sensitive, a cellular redox agent functionsas their regulator. Heo, J. (2011) Antioxid. Redox Signaling 14,689-724. Most Rho proteins, including Rac1 and Cdc42, are redoxsensitive because they possess a single redox-sensitive cysteine (Cys¹⁸,Rac1 numbering) in the GXXXXGK(S/T)C (SEQ ID NO:1) motif (monothiol).Heo, J., et al. (2005) J. Biol. Chem. 280, 31003-31010. A subset of theGXXXXGK(S/T)C (SEQ ID NO:1) motif is found in RhoA and RhoB. Heo et al.(2006) Biochemistry 45, 14481-14489. This subset contains a secondarycysteine (Cys¹⁶, RhoA numbering) in addition to the primaryredox-sensitive cysteine (Cys²⁰, RhoA numbering, which is equivalent tothe Rac1 Cys¹⁸) termed the GXXXCGK(S/T)C (SEQ ID NO:2) motif (dithiol).Although both the GXXXXGK(S/T)C (SEQ ID NO:1) and GXXXCGK(S/T)C (SEQ IDNO:2) motifs have the same redox sensitivity (Heo, J. (2005) J. Biol.Chem. 280, 31003-31010) the latter has an additional redox modulationfunction. Heo et al. (2006) Biochemistry 45, 14481-14489. An analysis ofthe RhoC crystal structure PDB 2GCO (Dias et al. (2007) Biochemistry 46,6547-6558) in conjunction with a sequence analysis indicates that RhoCalso possesses the GXXXCGK(S/T)C motif (SEQ ID NO:2). Ras GTPasescontain a distinct redox-sensitive NKCD (SEQ ID NO:3) motif. Lander, H.M. (1997) FASEB J. 11, 118-124. Furthermore, the RhoC Cys²⁰ site (theRhoC numbering is the same as the RhoA numbering) is located at the Rhonucleotide-binding site (Dias et al. (2007) Biochemistry 46, 6547-6558),but Ras Cys¹¹⁸ (Harvey Ras numbering) is remote from the Rasnucleotide-binding site. Pai et al. (1989) Nature 341, 209-214. VariousRab family GTPases also possess the GXXXXGK(S/T)C (SEQ ID NO:1) motif(e.g., Rab1, Rab1A, Rab1B, Rab2, Rab2A/B, Rab4, Rab4A/B, Rab8, Rab8A/B,Rab10, Rab13, Rab14, Rab15, Rab19, and Sec4). See Heo, J. (2011)Antioxid. Redox Signal. 14, 689-724.

6-Thiopurine (6-TP) prodrugs, including 6-thioguanine (6-TG),6-mercaptopurine, and azathioprine, are antimetabolites. They are widelyused to treat cancers such as acute lymphoblastic leukemia, acutemyeloid leukemia and adenocarcinomas, and autoimmune disorders such asinflammatory bowel disease, Crohn's disease and rheumatoid arthritis, aswell as to treat organ transplant recipients. Elion, G. B. (1989)Science 244, 41-47; Langmuir et al. (2001) Best Pract. Res. Clin.Haematol. 14, 77-93; Gearry et al. (2005) J. Gastroenterol. Hepatol. 20,1149-1157.

In cells, cellular enzymes convert inactive prodrug 6-TPs into thepharmacologically active 6-thioguanine nucleotide that can be groupedinto deoxy-6-thioguanosine phosphate (6-TdGNP) and the 6-thioguanosinephosphate (6-TGNP). Elion, G. B. (1989) Science 244, 41-47; Langmuir etal. (2001) Best Pract. Res. Clin. Haematol. 14, 77-93; Gearry et al.(2005) J. Gastroenterol. Hepatol. 20, 1149-1157; McDonald et al. (1996)Cancer Res. 56, 2250-2255; Tiede et al. (2003) J. Clin. Invest. 111,1133-1145; de Boer et al. (2007) Nat. Clin. Pract. Gastroenterol.Hepatol. 4, 686-694. Furthermore, depending on the number of ribosephosphates, 6-TGNP can be further classified as 6-thioguanosinediphosphate (6-TGDP) and triphosphate (6-TGTP). 6-TdGNP can beincorporated into the de novo synthesis of DNA as a form of 6-TG. 6-TGin DNA can then be recognized as a DNA lesion by the mismatch repairsystem, which results in induction of the mismatch repair-mediated cellapoptosis. Lage et al. (1999) J. Cancer Res. Clin. Oncol. 125, 156-165;Yan et al. (2003) Clin. Cancer Res. 9, 2327-2334; Karran, P. (2006) Br.Med. Bull. 79-80, 153-170. This 6-TG-mediated induction of mismatchrepair is believed to be the main mechanism for the action of 6-TPs inthe treatment of acute lymphoblastic leukemia.

In contrast to 6-TdGNP, neither the metabolic path of 6-TGNP nor itstherapeutic activity and/or cytotoxicity has been clearly established. Afew recent studies have addressed the action of 6-TGNP on small GTPases.It was recently shown that long-term treatment of Ras-activated tumorcells, such as bladder carcinoma (cell-line, T24) and fibrosarcoma(cell-line, HT1080), with 6-TG results in production of cellular 6-TGNPthat targets Ras GTPase. Heo et al. (2010) Biochemistry 49, 3965-3976.This Ras-targeting action of 6-TGNP results in downregulation of Ras,which in turn terminates the tumorous growth of these cells. ThisRas-targeting action of 6-TGNP extends beyond its effect on tumor cellsand is considered cytotoxic because it deregulates the Ras GTP/GDPcycle.

It also has been shown that 6-TGNP targets and inactivates Rho GTPasessuch as Rac1. Tiede et al. (2003) J. Clin. Invest. 111, 1133-1145; Poppeet al. (2006) J. Immunol. 176, 640-651. This Rho GTPase-targeting actionof 6-TGNP may be attributable to the therapeutic effect of 6-TPs on theimmune system as well as on inflammatory bowel disease. Cuffari et al.(1996) Can. J. Physiol. Pharmacol. 74, 580-585; Maltzman et al. (2003)J. Clin. Invest. 111, 1122-1124; Quemeneur et al. (2003) J. Immunol.170, 4986-4995. The therapeutic action of 6-TP in inflammatory boweldisease correlates with the Rac1-targeting 6-TGNP that blocks Rac1 GNEby its GEF Vav (Poppe et al. (2006) J. Immunol. 176, 640-651), althoughthe details of the molecular mechanism by which this occurs are unknown.

Various hormone-related agents and Rho GTPase-downstream effectorblockers, as anti-breast tumor drugs, are available. These availabledrugs are not Rho specific, and thus they also perturb manynoncancer-related cellular signaling transductions. However, becausethese cellular signaling events are vital for cell survival,inhibition/deregulation of these signaling cascades is normallycytotoxic. A desirable chemotherapeutic agent for RhoC-overexpressed IBCis needed that would target RhoC directly and inhibit it. Directtargeting of tumorigenic RhoC by such an agent could reduce cytotoxicitywhile maximizing the antitumor effect. Similarly, therapeutic agentsthat directly target and inhibit RhoA and Rac1 activity for treatingdisorders including blood vessel diseases and autoimmune diseases,respectively, also are needed Inhibitors of various Rab family GTPasesare also needed.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides compositions and methodsfor treating diseases or conditions associated with a redox-sensitiveGTPase, such as a Rho or Rab family GTPase, in a subject, includingdiseases characterized by overexpression and/or misregulation of a Rhoor Rab family GTPase. In accordance with the present invention, variousdiseases can be treated by inhibiting a Rho or Rab family GTPase usingeffective amounts of a redox-sensitive purine compound and an effectiveamount of a redox agent that enhances the inhibition of the Rho or RabGTPase by the redox-sensitive purine compound.

In another aspect, the invention provides a composition for inhibiting aredox-sensitive GTPase protein, including a Rho or Rab family GTPase.The composition comprises an effective amount of a redox-sensitivepurine compound and an effective amount of a redox agent. In someembodiments, the redox-sensitive purine compound is selected from thegroup consisting of a 6-thiopurine compound, including 6-thioguanine,and an 8-thiopurine compound, including 8-thioguanine. In someembodiments, the redox-sensitive purine is selected from the group ofcompounds having the general structure of formula I, wherein:

R₁ is selected from the group consisting of C6-thioxo (C6=S; incombination with C6 of the purine base); C6-thiol (C6-SH; in combinationwith C6 of the purine base); C6-selenal (C6=Se; in combination with C6of the purine base); C6-selenol (C6-SeH; in combination with C6 of thepurine base); C1-4 straight chain or branched chain alkyl, alkenyl, oralkynyl, wherein C1-4 are unsubstituted, singly substituted or multiplysubstituted, wherein the substituents are selected from the groupconsisting of thiol, thioxo, selenal, selenol, hydroxyl, halogen, amino,ketone, alkoxy, aldehyde and carboxylic acid; OH; and O;wherein R₂ is selected from the group consisting of H; thiol (SH);selenol (SeH); C1-4 straight chain or branched chain alkyl, alkenyl, oralkynyl, wherein C1-4 are unsubstituted, singly substituted or multiplysubstituted, wherein the substituents are selected from the groupconsisting of thiol, thioxo, selenal, selenol, hydroxyl, halogen, amino,ketone, alkoxy, aldehyde and carboxylic acid; NH₂; and OH;wherein R₃ is selected from the group consisting of H, NH₂ and OH;with the proviso that at least one of R₁ or R₂ is a moiety thatcomprises a redox-sensitive functional group selected from the groupconsisting of thioxo, thiol, selenal and selenol.

The redox agent may be selected from any number of possible agents,including, for example, nitric oxide, nitrogen dioxide, dinitrogentrioxide, superoxide anion radical, hydrogen peroxide, and carbonateradical. The redox agent may also be produced indirectly by anotheragent that stimulates the production of the redox agent.

In another aspect, the invention provides a method for treating adisease associated with a redox-sensitive GTPase protein, including aRho or Rab family GTPase protein, in a subject. The method comprisesadministering to the subject an effective amount of a redox-sensitivepurine compound and an effective amount of a redox agent. In someembodiments, the redox-sensitive purine compound is selected from thegroup consisting of a 6-thiopurine compound, including 6-thioguanine,and an 8-thiopurine compound, including 8-thioguanine. Otherredox-sensitive purine compounds may be selected from one of the groupsof compounds of formula I above. The administration of theredox-sensitive purine compound with the redox agent inhibits theredox-sensitive GTPase protein (e.g., the Rho or Rab family GTPase) andthe disease is treated.

Various diseases may be treated with the present invention. For example,various types and forms of cancer are treatable, including breast cancerand prostate cancer. In some embodiments, the invention provides amethod of treating prostate cancer, comprising administering aneffective amount of the redox-sensitive purine compound and an effectiveamount of the redox agent to the subject, wherein a redox-sensitiveGTPase is inhibited. In some embodiments, the redox-sensitive GTPase isa Rho family GTPase. In one embodiment, the Rho family GTPase is a Racprotein. In some embodiments, the redox-sensitive GTPase is a Rab familyGTPase. In some embodiments, the invention provides methods for treatingcancer metastasis in a subject, comprising administering an effectiveamount of a redox-sensitive purine compound and an effective amount of aredox agent, wherein a Rho family GTPase is inhibited to treat themetastasis. In one embodiment, the Rho family GTPase is RhoC. Theinvention further provides methods for treating various blood vesseldiseases, comprising administering an effective amount of aredox-sensitive purine compound and an effective amount of a redox agentto a subject, wherein a Rho or Rab family GTPase is inhibited to treatthe blood vessel disease. In one embodiment, the Rho family GTPase isRhoA. In some blood vessel diseases, for example, hypertension,inhibiting RhoA promotes vasorelaxation of blood vessels. The inventionfurther provides methods of treating immune related disorders, such asautoimmune disorders, comprising administering an effective amount of aredox-sensitive purine compound and an effective amount of a redoxagent, wherein a Rho or Rab family GTPase is inhibited to treat theimmune related disorder. In one embodiment, the Rho family GTPase isRac1.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary, and thus do notrestrict the scope of the invention.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1. (A) Classification of the Ras superfamily. (B) Small GTPaseactivity regulation cycle.

FIG. 2. Sequence alignment of Rho GTPases. A software ClustalX (version1.83) was used for multiple sequence alignment of Rho GTPases. Sequencealignments longer than #49 (Rac1 numbering) were omitted forconvenience, but the H and KRas sequences were shown for comparison.

FIG. 3. Spatial architecture of the Phe²⁸ side chain and theredox-sensitive motif of GTPases with the bound nucleotide. Theredox-active Rho family GTPases possess the GXXXXGK(S/T)C motif (SEQ IDNO:1) (A) instead of the NKCD (SEQ ID NO:3) motif that is found in theredox-active Ras family GTPases (B). The distance and spatialorientation of the Phe²⁸ side chain, nucleotide ligand, and redox-activethiol group in the GXXXXGK(S/T)C (SEQ ID NO:1) motif of Rac1 (A) and theNKCD motif of Rap1A (B) are presented. The Figure was generated by usingRASMOL with PDB 1MH1 for the GXXXXGK(S/T)C (SEQ ID NO:1)motif-containing Rac1 GTPase and PDB 1C1Y for the NKCD (SEQ ID NO:3)motif-containing Rap1A GTPase.

FIG. 4. Structures of guanine base and its analogs.

FIG. 5. Determination of cell invasiveness in the presence and absenceof 6-thiopurine and/or a redox agent. The top chamber of a Transwellfilter (6.5 mm with 8 μm pores, Costar; Corning, N.Y.) was coated with a10 μL aliquot of 10 mg/mL Matrigel (BD Biosciences, Bedford Mass.), andthe lower chamber of the Transwell was filled with either serum-free orserum-containing media. Sample cells (e.g., 6-TG-resistant HME-RhoCcells) were prepared to resuspend cells in a serum-free medium with 0.1%BSA at a concentration of 4×10⁵ cells/mL. At this point, neither6-thiopurine nor GSNO were added to the cells. The sample cells (0.5 mL)were added to the top chamber of a Transwell filter and were incubatedfor 24 h at 37° C. in a 10% CO2 incubator. The Transwell filters werefixed with methanol, stained with H&E, and cells in the serum-containingsamples were counted. The number of cells that had invaded theserum-free medium-containing lower chambers was used as a controlbackground. Results are given as percent invasion over that seen inHME-RhoC cells (100%) and represent mean and standard error values fromtriplicate measurements.

FIG. 6. Detection of unusual 6-TGNP adduct from HME-RhoC cells. HME-RhoCcells were treated with 6-TG (1 μM; every 48 h) for 5 days and werewashed twice in phosphate-buffered saline. A monoclonal RhoC antibody(Biocompare, South San Francisco, Calif.) and a “MammalianCo-Immunoprecipitation Kit” (Pierce) were used for the analyses of theRhoC 6-TGNP interaction in HME-RhoC cells. The immunoprecipitated RhoCsample was suspended in 50% methanol:0.1% formic acid. The proteinportion and RhoC-released nucleotide were separated by a briefcentrifugation (3000×g for 5 min). The protein portion from theimmunoprecipitated RhoC was resuspended in the assay buffer (pH 7.5),digested with trypsin for 10 h, and analyzed with MALDI-TOF tandem massspectrometry. The MALDI-TOF peak at 1476.6 Da represents peptide-6-TGDPadduct; TC²⁰LLIVFSK-6-TGDP (SEQ ID NO:4). (A) and (B), respectively,denote any of the RhoC samples taken from cell culture without and withtreatment of 6-TG (1 μM; every 48 h).

FIG. 7. Proposed mechanisms of the Rho 6-TGNP disulfide adductionformation. 6-TGNP is represented in blue. The dotted lines in blackrepresent putative hydrogen-bond interactions between RhoC residues and6-TGNP.

FIG. 8. Examination of Ras and Raf-1 activity in T24 cells treated withvarious reagents. (A) T24 cells were treated with 6-TG (1 μM, every 48h) and DETA/NO (10 μM; every 8 h) for 17 days, and total Ras expressionand its activity per cell mass was determined using an anti-HRas- and ananti-Raf-1-specific antibody, respectively, at the date indicated. (B)T24 cells were pretreated with 6-TG (1 μM; every 48 h) followed bytreatment with DETA/NO (10 μM; every 8 h) for a day at the indicateddate and analyzed for a Ras downstream effector Raf-1 activity usingGST-MEK1 as a Raf-1 substrate. The total Raf-1 expression also wasdetermined using an anti-Raf-1 antibody. Actin expression in cells wasshown as controls.

FIG. 9. Molecular weight determination of the .NO₂-mediated Ras-bound6-TGNP dissociation product by ESI. As noted elsewhere, T24 cellspossess oncogenic Ras G12V. Before treating the tumor cells with GSNO(10 μM; every 8 h) or DETA/NO (10 μM; every 20 h), cells treated with6-TG (1 μM; every 48 h) for 8 days were washed twice inphosphate-buffered saline. A monoclonal antibody for Ras (Sigma) and a“Mammalian Co-Immunoprecipitation Kit” (Pierce) were used for theanalyses of the Ras GTPase in T24 cells. The immunoprecipitated Rassample was suspended in 50% methanol:0.1% formic acid. The proteinportion and Ras-released nucleotide were separated by a briefcentrifugation (3000×g for 5 min). To determine whether the T24 cell Rasreleases chemically modified nucleotide(s), the supernatant containingRas-released nucleotide(s) from the immunoprecipitated Ras sample wasanalyzed by ESI-MS.

FIG. 10. Proposed mechanism of redox agent-mediated Ras 6-TGNPdissociation. 6-TGNP is represented in blue, and the NO₂ moiety of thenucleotide-nitration adduct is shown in red. The ribose phosphate moietyof 6-TGNP is depicted by R. The dotted lines in black represent putativehydrogen-bond interactions between Ras residues and 6-TGNP.

FIG. 11. Determination of cell invasiveness in the presence and absenceof 8-thiopurine and/or a redox agent. The invasion motility assay forHME-RhoC cells was performed as described essentially in FIG. 5, exceptthat 8-TG, instead of 6-TG, was used. The number of cells that hadinvaded the serum-free medium-containing lower chambers was used as acontrol background. Results are given as percent invasion over that seenin HME-RhoC cells (100%) and represent mean and standard error valuesfrom triplicate measurements.

FIG. 12. Spatial architecture of RhoC with the bound nucleotide. Thedistance and spatial orientation of the Phe³⁰ side chain, nucleotideligand, and the redox-sensitive thiolate group in the GXXXXGK(S/T)C (SEQID NO:1) motif of RhoC are shown. The distance between the sulfur atomof the Cys²⁰ side chain and the C8 carbon of GDP is estimated to be ˜3.6Å. The distance between the sulfur atom of the Cys²⁰ side chain and thesulfur atom of 8-TG base is shown to be ˜6.9 Å. Finally, the distancebetween the sulfur atom of the Cys²⁰ side chain and the sulfur atom of8-TG base is calculated to be ˜2.1 Å. The figure was generated by usingRASMOL with PDB 1GCO.

FIG. 13. Guanine nucleotide exchange of small GTPases with 6-TGDP. (A)To monitor 6-TGDP-mediated GTPase GDP dissociation, [³H]GDP-loadedGTPase (1 μM) was incubated with 6-TGDP (0-2000 μM) for 1 h. (B) Tomonitor GTPase 6-TGDP dissociation by GDP (0-2000 μM) in the presenceand absence of dithiothreitol (DTT), 6-TGDP-loaded GTPase (˜1 μM) wastreated with .NO₂ (˜3 μM) under anaerobic conditions and was incubatedwith various concentrations of [³H]GDP (0-2000 μM) in the presence andabsence of DTT (10 mM) for 1 h. For all assays, aliquots were withdrawnand spotted onto nitrocellulose filters. The filters were then washedthree times with an assay buffer, and radioactivity was determined usinga Beckman-Coulter scintillation counter. The resultant radioactivity(dpm) values associated with GTPase-bound [³H]GDP were converted intothe fraction of mol nucleotide per mol total GTPase. The data presentedrepresent mean and standard error values from triplicate measurements.The apparent dissociation constants (appKD values) of GTPase 6-TGDPassociation ([³H]GDP dissociation, A); and 6-TGDP dissociation ([3H]GDPassociation, B) were obtained by fitting the data to a simpledissociation and association, respectively. (A) The ^(app)KD values ofRas, RhoC, RhoA and Rac1 for 6-TGDP, are, respectively, 2300, 230, 224,and 245 μM [6-TGDP]. (B) The appKD values of RhoC with and without DTTare, respectively, 560, and 1000 μM [[³H]GDP], whereas the ^(app)KDvalues of Ras with and without DTT are 530, and 520 μM [[³H]GDP],respectively. The standard errors of each ^(app)KD value are less than10% of their given values, and regression values associated with the fitwere r²>0.9560.

FIG. 14. Determination of cell invasiveness in the presence and absenceof thiopurines and/or redox agents. (A) Experimental procedures for thedetermination of cellular RhoC expression in cells treated with 6-TGand/or DETA/NO, as well as for the determination of cell motility usingMatrigel and the corresponding RhoC activity of cells treated with 6-TGand/or DETA/NO, are described elsewhere herein. The average number andstandard error of triplicate measurements of SUM149 cells from invasionthrough the Transwell filter pores to the lower chambers in the absenceof 6-TG and DETA/NO were determined to be 130±4. All other cell invasionresults were normalized against the average value of the SUM149 cellinvasion that was set to be 100%, and represent average and standarderror values from triplicate measurements: a, cells untreated with 6-TGand DETA/NO; b, cells treated with only 6-TG; c, cells treated with onlyDETA/NO; and d, cells treated with both 6-TG and DETA/NO. Western blotanalysis representing the total expression of RhoC in these cells alsois shown. (B) Experimental procedures for the determination of thetime-dependent motility of cells using the QCM Cell Invasion Assay kitin the presence and absence of 6-TG and DETA/NO are described elsewhereherein. The determined maximal mean value of the optical density andstandard error of triplicate measurements corresponding to the invasionof SUM149 cells from the upper to the lower chamber through filter poreswithout treatment with 6-TG and DETA/NO at the day four of experimentwas estimated to be 1.13±0.16. All colorimetrically determined opticaldensity values associated with cell invasion under these experimentalconditions were normalized against the mean value of optical density forthe value of the SUM149 cell invasion in the absence of 6-TG and DETA/NOat the day four of experiment that was set to be 100%. All valuesrepresent mean and standard error values from triplicate measurements:SUM149 cells untreated (◯) or treated (●) with 6-TG and DETA/NO; SW480cells untreated (□) or treated (▪) with 6-TG and DETA/NO; and HCCLM3cells untreated (⋄) or treated (♦) with 6-TG and DETA/NO.

FIG. 15. Detection of unusual 6-TGNP adduct from SUM and HME-RhoC cells.The ESI-MS and MS/MS methods to detect and identify a chemicallymodified RhoC residue are described elsewhere herein. (A) A mass peak of1481.6 Da. was detected when SUM149 or HME-RhoC cells were treated with6-TG. Given that the ESI-MS sample was prepared with co-IP using theRhoC antibody from the 6-TG treated SUM149 or HME-RhoC cells followed bytrypsin digestion, the best candidate to give rise to the spectrum ofthe mass peak at 1481.6 Da. is a RhoC fragment TC²⁰LLIVFSK crosslinkedto 6-TGDP (TC²⁰LLIVFSK-6-TGDP disulfide adduct) (SEQ ID NO:4). The leftand right columns, respectively, denote any of the RhoC samples takenfrom SUM and HME cells. As indicated, a mixture of samples taken fromuntreated SUM149 and SUM102 cells was used as a control for theexperiment shown in the left column. For the right column, a mixture ofsamples taken from HME-RhoC and HME-C205 RhoC cells was used as acontrol. (B) A MS/MS analysis was performed with the ESI-MS sample of6-TG-treated SUM149 cells to identify a biomolecule that have a mass of1481.6 Da. Major MS/MS peaks shown were best fit to the masses of ionfragments of the TC²⁰LLIVFSK-6-TGDP adduct (SEQ ID NO:4): (i) the 1462.8Da. fragment, formed upon loss of a H₂O from the β-phosphate of the6-TGDP moiety of TC²⁰LLIVFSK-6-TGDP adduct (SEQ ID NO:4); (ii) the1445.6 Da. fragment, formed by losing a H₂O and a OH from the β- andα-phosphate of the 6-TGDP moiety of TC²⁰LLIVFSK-6-TGDP adduct (SEQ IDNO:4); (iii) the 1400.4 Da. fragment, formed upon loss of COOH from theC-terminus of TC²⁰LLIVFSK-6-TGDP adduct (SEQ ID NO:4) as well as a H₂Oand a OH from the β- and α-phosphate of the 6-TGDP moiety ofTC²⁰LLIVSK-6-TGDP adduct (SEQ ID NO:4); (iv) the 1022.8 Da. fragment,formed upon loss of the 6-TGDP moiety from TC²⁰LLIVFSK-6-TGDP adduct(SEQ ID NO:4); and (v) the 978.3 Da. fragment, formed by losing thepeptide C-terminus COOH and the 6-TGDP moiety from TC²⁰LLIVFSK-6-TGDPadduct (SEQ ID NO:4). Taking into consideration the MS/MS result underour experimental conditions (e.g., SUM149 cells treated with 6-TG), the1481.6 Da. molecule detected in the ESI-MS analysis is assigned to bethe TC²⁰LLIVFSK-6-TGDP disulfide adduct (SEQ ID NO:4). *Unassigned masspeaks.

FIG. 16. Kinetic properties of RhoC with 6-TGDP in the presence orabsence of a redox agent. All kinetic analyses are described elsewhereherein. (A) ^(app)K_(D) values of Rac1, RhoA, RhoC, and RhoC C20S for6-TGDP were estimated to be 7.9, 11.6, 11.8, and 11.2 μM, respectively.^(true)K_(D) values of Rac1, RhoA, and RhoC for 6-TGDP were thencalculated to be 5.1, 10.8, 11.1, and 10.5 μM, respectively, by using acompensation equation, ^(true)K_(D) for 6-TGDP=^(app)K_(D) for6-TGDP/(1+[[³H]GDP]/^(true)K_(D) for GDP) (Heo, J., and Hong, I. (2010)Biochemistry 49, 3965-3976), in conjunction with the values given. (The^(true)K_(D) values of Rac1 and RhoA for GDP were known to be 1.8 μM and13.0 μM (Morgan et al. Cancer Res. 54, 5387-5393 (1994)). Within thisstudy, the ^(true)K_(D) value of RhoC and RhoC C20S for GDP wasdetermined to be 15.0±8 μM and 14.2±8 μM, respectively.) (B) The ratesof GDP dissociation from RhoC by NO, .NO₂, and N₂O₃ (a mixture of NO and.NO₂) were determined to be 0.19×10⁻³, 6.04×10⁻³, and 0.21×10⁻³ s⁻¹,respectively. The GDP dissociation rates from RhoC C20S by NO, .NO₂, andN₂O₃ were estimated to be 0.02×10⁻³, 0.05×10⁻³, and 0.04×10⁻³ s⁻¹,respectively. (C) The NO-, .NO₂-, and N₂O₃-mediated rates of 6-TGDPdissociation from RhoC by NO, .NO₂, and N₂O₃ were determined to be0.08×10⁻³, 2.65×10⁻³, and 0.16×10⁻³ s⁻¹, respectively. The rates of6-TGDP dissociation from RhoC C20S in the presence of NO, .NO₂, and N₂O₃were estimated to be 0.04×10⁻³, 6.27×10⁻³, and 0.12×10⁻³ s⁻¹,respectively. (D) ^(app)K_(D) values of RhoC for [³H]GDP in the presenceor absence of DTT were estimated to be 22.4 μM and 103.7 nM,respectively. ^(app)K_(D) values of C20S RhoC for [³H]GDP in thepresence or absence of DTT were determined to be 24.8 μM and 25.2 μM,respectively. The regression values associated with these fitswere >0.8595. All data points associated with vertical standard errorbars shown in this figure are the mean values of triplicatemeasurements.

FIG. 17. Proposed mechanisms of the Rho 6-TGNP disulfide adductionformation. A symbol M in red represents a transition metal, serving foran electron acceptor. The dotted lines represent putative hydrogen-bondinteractions between RhoC residues and 6-TGNP.

FIG. 18. Proposed mechanisms of action of 6-TGNP and a redox agent onRas GTPases.

FIG. 19. Spatial architecture of the Phe²⁸ side chain and the NKCD motifof GTPases with the bound nucleotide. The redox-active Rho familyGTPases possess the GXXXXGK(S/T)C (SEQ ID NO:1) motif (B) instead of theNKCD (SEQ ID NO:3) motif that is found in the redox-active Ras familyGTPases (A). The distance and spatial orientation of the Phe²⁸ sidechain, nucleotide ligand, and redox-active thiol group in the NKCD andGXXXXGK(S/T)C (SEQ ID NO:1) motif of Rap1A (A) and Rac1 (B),respectively, are presented. The Figure was generated by using RASMOLwith PDB 1C1Y for the NKCD (SEQ ID NO:3) motif-containing Rap1A GTPaseand PDB 1MH1 for the TKLD motif-containing Rac1 GTPase.

FIG. 20. Determination of RhoA activity and MLC phosphorylation. A7r5cells treated with 6-TG (1 μM) and/or DETA/NO (10 μM) for 3 days. Celllysates in a buffer containing 1% NP-40, 10 mM EDTA, and 20 mM TrisHCl(pH 7.4) were centrifuged (50,000×g, 20 min) to remove solid matter. (A)The soluble cell extract was then used to perform Western blot analysesfor RhoA and the MLC. This cell extract also was used for theco-IP-based ESI-MS analysis (FIG. 6). (B) A RhoA activity assay wascolorimetrically determined using cell extract; the assay was performedwith the RhoA G-LISA Activation Assay Kit (Cat. No. BK124) (Denver,Colo.). To examine the level of phosphorylation of the MLC, cells werecultured with ˜5 μCi of ³²P in 1 mL of phosphate-free DMEM. Thecollected cell extract was nutated with resin coupled with a macrophagemyosin II antibody at 4° C. for 5 h. Resin was collected bycentrifugation, and proteins bound to the resin were washed and elutedwith a buffer (80° C.) containing 25% SDS, 5 mM DTT, and 10 mM TrisHCl(pH 6.8). The immunoprecipitates were then analyzed by SDS-PAGE andautoradiographed. The values of RhoA activity and the MLCphosphorylation determined from untreated cells were set at 100%. Alldata obtained from cells treated with 6-TG and/or DETA/NO were thenexpressed as normalized values against the standards initiallyestablished. The data represented in the figure represent the meanvalues of triplicate measurements, and the vertical error bar indicatesstandard errors.

FIG. 21. Detection of 6-TGNP adduct from A7r5 and T cells. Experimentalprocedures using A7r5 (A and B) and T (C and D) cells, respectively,were identical to those of the experiments described in FIG. 19 andprevious studies (Tiede et al. J. Clin. Invest. 111, 1133-1145 (2003);and Poppe et al. J. Immunol. 176, 640-651 (2006)). The cell extract wasincubated with a monoclonal RhoA antibody (A and B) or a Rac1 antibody(C and D) coupled with resin. Resin was collected by centrifugation(5,000×g, 10 min), which was suspended in 50% methanol:0.1% formic acid.The protein portion was separated by brief centrifugation (3,000×g for 5min), resuspended in a buffer containing 10 mM EDTA and 5 mM phosphate(pH 7.5), digested with trypsin for 10 h, and analyzed with MS. The MSpeak at 1476.6 Da represents peptide-6-TGDP adduct; TC²⁰LLIVFSK-6-TGDP.

FIG. 22. Detection of 8-TGNP adduct from A7r5 cells. Experimentalprocedures using A7r5 cells were identical to those of the experimentsdescribed in FIG. 19, except that 8-TG instead of 6-TG was used. Theindicated MS peak at 1476.6 Da represents peptide-8-TGDP adduct;TC²⁰LLIVFSK-8-TGDP (SEQ ID NO:4). The molecular weight of thepeptide-8-TGDP adduct is exactly the same as that of the peptide-6-TGDPadduct. A and B, respectively, represent any of samples taken from acell culture with and without treatment with 8-TG.

FIG. 23. Mechanism of pulmonary vasocontraction and dilation. The solidarrows indicate the paths of activation, as well as show activation andinactivation of cellular proteins. A dotted arrow shows an NO mediatedstimulation path of sGC. Abbreviations: ET-1, endothelin-1; PLC,phospholipase C; IP3, inositol trisphosphate; CaM, Calmodulin; NO,nitric oxide; sGC, soluble guanylyl cyclase; cGMP, cyclic GMP; PKG, cGMPdependent protein kinase; MLCK, myosin light chain kinase; ROCK, RhoGEF,Rho guanine nucleotide exchange factor; Rho kinase; MLCP, myosin lightchain phosphatase; and MLC, myosin light chain. MLCP consists of threedomains as indicated with a dotted line: MBS (also known as MYPY-1), amyosin-binding subunit; PP1, a catalytic domain; and an *unknownnoncatalytic domain.

FIG. 24. Effects of 6- and 8-TG on A7r5 cells. Treatment of cells with6- or 8-TG is described in FIGS. 19 and 21. The image associated withthe effect of 6- or 8-TG was taken after 5 days of treatment.

FIG. 25. Effect of 6-TG in combination with DETA/NO on the viability andcaspase activity of cells. MTT and caspase assays for cells treated with6-TG (1 μM at every 24 h) and/or DETA/NO (10 μM at every 20 h) wereperformed as described elsewhere herein. (A) MTTassay: for each cellline, the determined values of cell viability in the presence of 6-TGand/or DETA/NO were normalized against the mean value of cell viabilityin the absence of 6-TG and DETA/NO (set to be 100%). (B) Caspase-3/8assay: the apoptotic activity of sample cells treated with the apoptoticinducer was set to be 100%, and other results were then expressed asnormalized values against the mean value associated with the treatmentwith the apoptotic inducer. For both (A) and (B), all values representthe mean values and standard error values from triplicate measurements.

FIG. 26. Determination of Rac1 activity and ERM phosphorylation.Activated T cells treated with 6-TG (1 μM) and/or DETA/NO (10 μM) for 3days. Cell lysates in a buffer containing 1% NP-40, 10 mM EDTA, and 20mM TrisHCl (pH 7.4) were centrifuged (50,000×g, 20 min) to remove solidmatter. (A) The soluble cell extract was then used to perform Westernblot analyses for Rac1 and the ERM. This cell extract also was used forthe co-IP-based ESI-MS analysis (FIG. 8). (B) A Rac1 activity assay wascolorimetrically determined using cell extract; the assay was performedwith the Rac1 G-LISA Activation Assay Kit (Cat. No. BK125) (Denver,Colo.). To examine the level of phosphorylation of the ERM, cells werecultured in complete RPMI 1640 medium with ˜50 μCi of ³²P/mL beforeinitiating stimulation of T cells. The collected cell extract wasnutated with resin coupled with a human ERM (231C2a) antibody (SantaCruz Biotechnology, CA) at 4° C. for 5 h. Resin was collected bycentrifugation, and proteins bound to the resin were washed and elutedwith a buffer (80° C.) containing 25% SDS, 5 mM DTT, and 10 mM TrisHCl(pH 6.8). The immunoprecipitates were then analyzed by SDS-PAGE andautoradiographed. The value of Rac1 activity determined from untreatedcells was set at 100%. The value of the ERM phosphorylation determinedfrom cells treated with both 6-TG and DETA/NO was set at 100%. All dataobtained from cells treated with 6-TG and/or DETA/NO were then expressedas normalized values against the standards initially established. Thedata represented in the figure represent the mean values of triplicatemeasurements, and the vertical error bar indicates standard errors.

FIG. 27. Detection of 8-TGNP adduct from T and A7r5 cells. Experimentalprocedures using T (A and B) and A7r5 (C and D) cells were identical tothose of the experiments described in FIG. 8, except that 8-TG insteadof 6-TG was used. The indicated MS peak at 1476.6 Da representspeptide-8-TGDP adduct; TC²⁰LLIVFSK-8-TGDP (SEQ ID NO:4). The molecularweight of the peptide-8-TGDP adduct is exactly the same as that of thepeptide-6-TGDP adduct.

FIG. 28. Spatial architecture of Rac1 with the bound nucleotide. Thedistance and spatial orientation of the Phe28 side chain, nucleotideligand, and the redox-sensitive thiolate group in the GXXXXGK(S/T)C (SEQID NO:1) motif of Rac1 are shown. The distance between the sulfur atomof the Cys¹⁸ side chain and the C8 carbon of GDP is estimated to be ˜3.6Å. The distance between the sulfur atom of the Cys¹⁸ side chain and thesulfur atom of the 8-TG base is shown to be ˜6.9 Å. Finally, thedistance between the sulfur atom of the Cys¹⁸ side chain and the sulfuratom of the 8-TG base is calculated to be ˜2.1 Å. The figure wasgenerated by using RASMOL with PDB 1MH1.

FIG. 29. Model mechanism of 6-TP-mediated immunosuppression. CD28co-stimulation activates the Rac1 GEF Vav, which in turn enhancesreplacement of the Rac1-bound GNP with 6-TGNP (Path A). A redox agent,either .NO₂ or O₂.⁻ from NOS and NAD(P)H oxidase, respectively, triggersa disulfide bond formation between the Cys¹⁸ sulfur atom of Rac1 and thesulfur atom of 6-TGNP to produce a Rac1-6-TGNP disulfide adduct (PathB). Due to the disulfide bond, the Vav-mediated Rac1 GNE is blocked(Path C), resulting in accumulation of an inactive Rac1-6-TGNP disulfideadduct. Accumulation of inactive Rac1 GTPases leads to blockage of ERMdephosphorylation (Path D) that suppresses T cell activation. Dottedarrows show the pathway for the accumulation of 6-TGNP in cells.Abbreviations: CD28, Cluster of Differentiation 28; 6-TP, 6-thiopurine;6-TG, 6-thioguanine; 6-MP, 6-mercaptopurine; 6-TGNP, 6-thioguaninenucleotide; GNP, guanine nucleotide; NOS, Nitric Oxide Synthase; andERM, Ezrin-Radixin-Moesin.

DETAILED DESCRIPTION

The present invention is based on the discovery that redox-sensitivepurine compounds are capable of inhibiting the activity of Rho or Rabfamily GTPases in a redox-dependent manner. The Rho or Rab-targetingaction of the redox-sensitive purine compounds is enhanced by thepresence of a redox agent. Without being bound by theory, it is believedthat the purine compound forms an adduct with the Rho or Rab GTPase,preventing GTP binding and thereby inactivating the Rho or Rab GTPase.In accordance with some of the embodiments as described herein,compositions comprising an effective amount of a redox-sensitive purinecompound in combination with an effective amount of a redox agent areprovided for administration and use in inhibiting Rho or Rab GTPases. Insome embodiments, the compositions can be used to treat diseasesassociated with redox-sensitive GTPases such as Rho or Rab familyGTPases, including cancer, blood vessel diseases, and immune relateddiseases.

Reference will now be made in detail to embodiments of the inventionwhich, together with the drawings and the following examples, serve toexplain the principles of the invention. These embodiments describe insufficient detail to enable those skilled in the art to practice theinvention, and it is understood that other embodiments may be utilized,and that structural, biological, and chemical changes may be madewithout departing from the spirit and scope of the present invention.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the exemplary methods,devices, and materials are now described.

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found, for example, in Benjamin Lewin, Genes VII, published by OxfordUniversity Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); TheEncyclopedia of Molecular Biology, published by Blackwell Publishers,1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by Wiley,John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technicalreferences.

For the purpose of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa. In the event thatany definition set forth below conflicts with the usage of that word inany other document, including any document incorporated herein byreference, the definition set forth below shall always control forpurposes of interpreting this specification and its associated claimsunless a contrary meaning is clearly intended (for example in thedocument where the term is originally used). The use of “or” means“and/or” unless stated otherwise. The use of “a” herein means “one ormore” unless stated otherwise or where the use of “one or more” isclearly inappropriate. The use of “comprise,” “comprises,” “comprising,”“include,” “includes,” and “including” are interchangeable and notintended to be limiting. Furthermore, where the description of one ormore embodiments uses the term “comprising,” those skilled in the artwould understand that, in some specific instances, the embodiment orembodiments can be alternatively described using the language“consisting essentially of” and/or “consisting of.”

As used herein “about” means±10% of the indicated value.

The terms “Rho GTPases” or “Rho family” refer to a subfamily of the Rassuperfamily and are small, membrane-bound, Ras-related GTP-bindingproteins that function by binding and hydrolyzing GTP. Rho GTPasesfunction as molecular switches, cycling between an inactive GDP-boundconformation and an active GTP-bound conformation and includes, at leastRhoA, RhoB, RhoC, RhoG, Rac1, Rac2, Rac3, Cdc42, TC¹⁰, TCL. The term“Rab GTPase” also refers to a subfamily of the Ras superfamily andincludes, at least Rab1, Rab1A, Rab1B, Rab2, Rab2A/B, Rab4, Rab4A/B,Rab8, Rab8A/B, Rab10, Rab13, Rab14, Rab15, Rab19 and Sec4.

The terms “Rac GTPase” or “Rac” refer to Rac1, Rac2, and/or Rac3.

Purines are a class of heterocyclic aromatic organic compoundscomprising a pyrimidine ring fused to an imidazole ring. Purines includesuch compounds as purine, adenine, guanine, xanthine, hypoxanthine,isoguanine and uric acid. In accordance with the present invention, a“redox-sensitive purine” is a purine compound that is capable uponadministration of inhibiting a Rho or Rab family protein in a redoxdependent-manner.

Without being bound by theory, it is believed that the redox-sensitivepurine is converted to an active compound, perhaps inside the cell,which in turn reacts with a redox-sensitive cysteine residue of the Rhoor Rab family member, forming an adduct and inhibiting nucleotideexchange of the Rho or Rab family protein. A redox-sensitive cysteineresidue (C) is found in the following motif of various Rho and Rabfamily proteins: GXXXXGK(S/T)C (SEQ ID NO:1) (FIG. 2). Rho proteins canbe classified into two groups: (i) GXXXXGK(S/T)C (SEQ ID NO:1)motif-containing Rho GTPases (e.g., Rac1, Cdc42 and RhoG) (monothiol),which possess only one redox-active cysteine (Cys¹⁸, Rac1 numbering;equivalent to RhoA Cys²⁰); and (ii) GXXXCGK(S/T)C (SEQ ID NO:2)motif-containing Rho GTPases (RhoA, RhoB, and RhoC), which have tworedox-active cysteines (Cys²⁰, the primary; and Cys¹⁶, the secondarycysteine; RhoA numbering) (dithiol). The redox-sensitive purine that canbe used in accordance with the present invention is not limiting. Insome embodiments, the redox-sensitive purine compound is a thiopurinecompound (TP). In some embodiments, the redox-sensitive purine is aguanine analog. In some embodiments, the redox-sensitive purine issubstituted at the C6 position of the purine base with a moiety thatcomprises a redox-sensitive functional group. In some embodiments, theredox-sensitive purine is substituted at the C8 position of the purinebase with a moiety comprising a redox-sensitive functional group. Insome embodiments, the redox-sensitive functional group is selected fromthe group consisting of thiol, thioxo, selenal and selenol andcombinations thereof. In some embodiments, the redox-sensitive purine isa 6-thiopurine compound, such as 6-thioguanine. In some embodiments, theredox-sensitive purine is an 8-thiopurine compound, such as8-thioguanine. In some embodiments, the redox-sensitive purine isselected from the group of compounds having the general structure offormula I, wherein:

R₁ is selected from the group consisting of C6-thioxo (C6=S; incombination with C6 of the purine base); C6-thiol (C6-SH; in combinationwith C6 of the purine base); C6-selenal (C6=Se; in combination with C6of the purine base); C6-selenol (C6-SeH; in combination with C6 of thepurine base); C1-4 straight chain or branched chain alkyl, alkenyl, oralkynyl, wherein C1-4 are unsubstituted, singly substituted or multiplysubstituted, wherein the substituents are selected from the groupconsisting of thiol, thioxo, selenal, selenol, hydroxyl, halogen, amino,ketone, alkoxy, aldehyde and carboxylic acid; OH; and O;wherein R₂ is selected from the group consisting of H; thiol (SH);selenol (SeH); C1-4 straight chain or branched chain alkyl, alkenyl, oralkynyl, wherein C1-4 are unsubstituted, singly substituted or multiplysubstituted, wherein the substituents are selected from the groupconsisting of thiol, thioxo, selenal, selenol, hydroxyl, halogen, amino,ketone, alkoxy, aldehyde and carboxylic acid; NH₂; and OH;wherein R₃ is selected from the group consisting of H, NH₂ and OH;with the proviso that at least one of R₁ or R₂ is a moiety thatcomprises a redox-sensitive functional group selected from the groupconsisting of thioxo, thiol, selenal and selenol.

In some embodiments, the redox-sensitive purine can include8-thioguanine (2-amino-8-mercapto-1H-purin-6(9H)-one),8-methylthioguanine (2-amino-8-(mercaptomethyl)-1H-purin-6(9H)-one),7-methylthioguanine(2-amino-7-(mercaptomethyl)-8,9-dihydro-1H-purin-6(H)-one), and7-thioguanine (2-amino-7-mercapto-8,9-dihydro-1H-purin-6(H)-one). 7- and8-thioguanines and other purine analogs are commercially available(Azopharma Drug Development Services, Miramar, Fla.). 8-thiopurinesincluding 8-thioguanine is commercially available from USBiological:http://www.usbio.net/item/A1378-06.

In some embodiments, the redox-sensitive purine can include6-thiopurine, 8-thiopurine, and analogs thereof. Commercially available6-thiopurines include azathioprine (AZA) (Imuran® sold byGlaxoSmithKline and available as a generic); 6-mercaptopurine (6-MP)(Purinethol® sold by Teva and available as a generic); 6-thioguanine(6-TG) (Tabloid® sold by GlaxoSmithKline and available as a generic).AZA and 6-MP are prodrugs converted into 6-thioguanine in cells, whichis then converted into its active form 6-thioguanine nucleotide(6-TGNP). 8-thiopurine can be cellularly converted into its active form8-thioguanine nucleotide (8-TGNP)

Also included within the scope of the redox-sensitive purine compoundsare pharmaceutically acceptable derivatives. By “a pharmaceuticallyacceptable derivative” is meant any pharmaceutically orpharmacologically acceptable salt, ester or salt of such ester of acompound according to the invention. The redox-sensitive purinecompounds may be converted into a pharmaceutically accepted ester byreaction with an appropriate esterifying agent, e.g. an acid halide oranhydride. The redox-sensitive purine compounds including estersthereof, may be converted into pharmaceutically acceptable salts thereofin conventional manner, e.g. by treatment with an appropriate acid. Anester or salt of an ester may be converted into the parent compound,e.g. by hydrolysis.

In some embodiments, the redox-sensitive purine is capable of inhibitinga Rho family member selected from the group consisting of RhoA, RhoB,RhoC, RhoG, Rac1, Rac2, Rac3, Cdc42, TC¹⁰, TCL, and combinationsthereof. In other embodiments, the redox-sensitive purine is capable ofinhibiting a Rab family member selected from the group consisting ofRab1, Rab1A, Rab1B, Rab2, Rab2A/B, Rab4, Rab4A/B, Rab8, Rab8A/B, Rab10,Rab13, Rab14, Rab15, Rab19, Sec4 and combinations thereof.

The redox agent that can be used in accordance with the invention is notlimiting, provided, however, that it is capable of enhancing theinhibition of a Rho or Rab family protein when used in conjunction witha redox-sensitive purine. In some embodiments, the redox agent isselected from a reactive oxygen species (ROS) or a reactive nitrogenspecies (RNS). In some embodiments, the redox agent is selected form thegroup consisting of nitric oxide (NO), nitrogen dioxide (.NO₂),nonradical higher oxides such as dinitrogen trioxide (N₂O₃), superoxideanion radical (O₂.⁻), carbonate radical, hydrogen peroxide, hydroxylradical and combinations thereof. The redox agent also may be producedindirectly by another agent that stimulates the production of the redoxagent. Accordingly, these other agents are also construed as “redoxagents” as used herein. Generally, any redox agent is useful in theinvention that facilitates formation of a disulfide bond or adductbetween Rho or Rab proteins and the active form of the redox-sensitivepurine compound, including 6- and 8-thioguanine compounds.

In some embodiments, nitric oxide releasing agents are used, eitherdirectly or indirectly by administering compounds that stimulatecellular nitric oxide releasing agents. Direct nitric oxide releasingagents (also called NO donors) include: peroxynitrite; sodiumnitroprusside (SNP) (brand name: Nitropress); diethylenetriamine nitricoxide adduct (DETA/NO); S-Nitrosoglutathione (GSNO); S-nitrosothiolssuch as S-nitroso-glutathione, S-nitroso-N-acetylpenicillamine,S-nitroso-albumin, S—NO—N-acety-L-cysteine, S—NO-diclofenac; nitricoxide-releasing aspirins; S-nitroso nonsteroidal antiinflammatory drugs(S-NITROSO NSAIDs); nitric oxide releasing NSAIDs; 2-acetoxybenzoate2-(2-nitroxy-methyl)-phenyl ester (NCX-4016) and 2-acetoxybenzoate2-(2-nitroxy)-butyl ester; phosphodiesterase inhibitors (e.g.,sildenafil); and organic nitrate and nitrite esters, such asnitroglycerin, amyl nitrite, isosorbide dinitrate, isosorbide5-mononitrate, and nicorandil. See also Napoli et al. (2003) Annu. Rev.Pharmacol. Toxicol. 43, 97-123, describing nitric oxide-releasing drugs.Indirect drugs that stimulate cellular nitric oxide releasing agentsinclude: drugs such as 1,4-dihydropyridine calcium channel blockers(CCBs) that stimulate nitric oxide synthase (NOS) to produce cellularnitric oxide; ACE inhibitors; ANGII Type 1 receptor antagonists;statins; and drugs that stimulate cyclooxygenase-1 (COX-1) and/orcyclooxygenase-2 (COX-2) to produce cellular superoxide anion radical,which also facilitates disulfide bond formation.

In some embodiments, the invention provides compositions comprising aneffective amount of a redox-sensitive purine compound and an effectiveamount of a redox agent. As used herein an “effective amount” of aredox-sensitive purine is any amount which, when administered, inhibitsRho or Rab family GTPase activity. The activity can be modulated in acell, a tissue, a whole organism, in situ, in vitro (test tube, a solidsupport, etc.), in vivo, or in any desired environment. As used herein,an “effective amount” of a redox agent is any amount which, when used inconjunction with a redox-sensitive purine, enhances the inhibition ofRho or Rab family GTPase activity.

In some embodiments, the composition comprises an effective amount of aredox-sensitive purine compound and an effective amount of a redox agentin further combination with one or more therapeutic agents for thetreatment of the diseases or conditions as described herein.

The redox-sensitive purine compound according to the invention incombination with the redox agent may be administered for therapy by anysuitable route including oral, rectal, nasal, topical (includingtransdermal, buccal and sublingual), vaginal and parenteral (includingsubcutaneous, intramuscular, intravenous and intradermal). It will beappreciated that the preferred route will vary with the condition andage of the recipient, the nature of the disease to be treated and theredox-sensitive purine and redox agent.

In some embodiments, a suitable dose of the redox-sensitive purinecompound for each of the conditions described herein will be in therange of about 0.01 to about 250 mg per kilogram body weight of therecipient (e.g. a human) per day. In some embodiments, the dose is inthe range of about 0.1 to about 100 mg per kilogram body weight per dayand in other embodiments, in the range of about 1.0 to about 20 mg perkilogram body weight per day. In some embodiments, the dose is in therange of about 0.5±5.0 mg/kg/day, about 0.5±2.5 mg/kg/day, or about0.5±1.5 mg/kg/day. Unless otherwise indicated, all weights of theredox-sensitive purine are calculated as the parent compound; for saltsor esters thereof, the weights would be increased proportionally. Insome embodiments, the desired dose is presented as two, three, four,five, six or more sub-doses administered at appropriate intervalsthroughout the day. These sub-doses may be administered in unit dosageforms, for example, containing 5 to 150 mg, 10 to 100 mg, or 25 to 50 mgof redox-sensitive purine per unit dosage form.

In some embodiments, the redox-sensitive purine is administered toachieve peak plasma concentrations of from about 0.025 to about 100 μM,in some embodiments, about 0.1 to about 70 μM, or about 0.25 to about 50μM. In some embodiments, this may be achieved, for example, by theintravenous injection of a 0.1 to 5% solution of the redox-sensitivepurine, optionally in saline, or orally administered as a boluscontaining about 0.1 to about 250 mg/kg of the redox-sensitive purine.In some embodiments, desirable blood levels may be maintained by acontinuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or byintermittent infusions containing about 0.1 to about 15 mg/kg of theredox-sensitive purine.

The redox agent may be administered at any suitable dosage, and in someembodiments may be administered at a dosage such that a cellularconcentration of from about 0.5 μM to about 10 μM is achieved. In someembodiments, the cellular concentration of redox agent is about 3 μM.

While it is possible for the redox-sensitive purine and the redox agentto be administered alone it is preferable that they be administered as apharmaceutical formulation. In some embodiments, the formulations of thepresent invention comprise the redox-sensitive purine in combinationwith the redox agent, as defined above, together with one or moreacceptable carriers thereof and optionally other therapeutic agents. Insome embodiments, each carrier must be “acceptable” in the sense ofbeing compatible with the other ingredients of the formulation and notinjurious to the patient. Formulations include those suitable for oral,rectal, nasal, topical (including transdermal buccal and sublingual),vaginal or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. Such methods include the stepof bringing into association the active ingredients with the carrierwhich constitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then if necessary shaping the product.

Compositions suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the redox-sensitive purine in combination with theredox agent 1) in an optionally buffered, aqueous solution or 2)dissolved and/or dispersed in an adhesive or 3) dispersed in a polymer.A suitable concentration of the active compounds is about 1% to 25%, orabout 3% to 15%.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient(s); as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient(s) may also bepresented as a bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycollate, cross-linked povidone, cross-linked sodium carboxymethylcellulose) surface-active or dispersing agent. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein using, for example,hydroxypropylmethyl cellulose in varying proportions to provide thedesired release profile. Tablets may optionally be provided with anenteric coating, to provide release in parts of the gut other than thestomach.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient(s) in a flavored basis,usually sucrose and acacia or tragacanth; pastilles comprising theactive ingredient in an inert basis such as gelatin and glycerin, orsucrose and acacia; and mouthwashes comprising the active ingredient(s)in a suitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient(s) such carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous isotonic sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multidose sealed containers, for example, ampules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of an active ingredient(s).

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example, those suitable for oral administration mayinclude such further agents as sweeteners, thickeners and flavoringagents.

The present invention further provides methods of inhibiting aredox-sensitive GTPase, including a Rho or Rab family GTPase, comprisingadministering an effective amount of a redox-sensitive purine compoundand an effective amount of a redox agent. In some embodiments, theGTPase is present inside a cell, including in a whole organism, tissue,organ or cell culture system. In some embodiments, the GTPase isinhibited in vitro or in a cell-free system.

The present invention further provides methods of treating certaindiseases or conditions which are associated with redox-sensitiveGTPases, including Rho or Rab family GTPases, in a subject, includingconditions characterized by overexpression and/or misregulation of Rhoor Rab family GTPases.

As used herein, “treatment,” “treating” or “treat” includes boththerapeutic and prophylactic treatments. Accordingly, the compounds andcompositions can be used at very early stages of a disease, or beforeearly onset, or after significant progression.

For example, in some embodiments, the invention provides methods oftreating a disease or condition associated with a Rho or Rab familyGTPase in a subject in need of treatment, comprising administering tothe subject one or more compositions of the invention.

In some embodiments, the diseases or conditions to be treated areassociated with a Rho family GTPase selected from the group consistingof RhoA, RhoB, RhoC, RhoG, Rac1, Rac2, Rac3, Cdc42, TC¹⁰, TCL andcombinations thereof. In other embodiments, the diseases or conditionsto be treated are associated with a Rab family GTPase selected from thegroup consisting of Rab1, Rab1A, Rab1B, Rab2, Rab2A/B, Rab4, Rab4A/B,Rab8, Rab8A/B, Rab10, Rab13, Rab14, Rab15, Rab19, Sec4, and combinationsthereof.

In some embodiments, the invention provides methods of treating adisease or condition associated with a Rho or Rab family GTPase in asubject in need of treatment, comprising administering to the subject aneffective amount of a redox-sensitive purine compound and an effectiveamount of a redox agent. In some embodiments, the redox agent enhancesthe formation of an adduct between a redox-sensitive residue of a Rho orRab family GTPase and the active form of the redox-sensitive purinecompound, thereby inhibiting the Rho or Rab GTPase. In some embodiments,the Rho or Rab GTPase harbors a GXXXXGK(S/T)C (SEQ ID NO:1) motif thatis redox-sensitive. In some embodiments, the Rho or Rab GTPase harbors aGXXXCGK(S/T)C (SEQ ID NO:2) motif that is redox-sensitive.

In some embodiments, the subject to be treated is a mammal. Mammals thatcan be treated in accordance with the invention, include, but are notlimited to humans, dogs, cats, horses, mice, rats, guinea pigs, sheep,cows, pigs, monkeys, apes and the like.

The redox-sensitive purine compound and the redox agent can beadministered simultaneously, in either separate or combinedcompositions, or at different times, e.g. sequentially such that acombined effect is achieved.

In some embodiments, the disease or condition to be treated is cancer.In some embodiments, the disease or condition to be treated is cancermetastasis. In some embodiments, the cancer or cancer metastasis isselected from the group consisting of prostate cancer, such ashigh-grade prostatic intraepithelium neoplasia (HG-PIN), breast cancer,including inflammatory breast cancer (IBC), ovarian cancer, pancreaticcancer, lung cancer, gastric cancer, hepatocellular carcinoma, bladdercancer, colorectal cancer and cutaneous melanoma. In some embodiments ofthe methods for treating cancer, the Rho family GTPase that is inhibitedis RhoC. For example, RhoC has been shown to be overexpressed in severaltumors such as prostate, breast, ovarian, lung, pancreas, and gastriccancer as well as in bladder, colorectal and cutaneous melanoma,although other cancer-generating Ras GTPases are not overexpressed. cDNAarray analyses have also implicated overexpress ion of RhoC in amelanoma model of metastasis.

In some embodiments, various thiopurine drugs and a redox agenteffectively inhibit cancer metastasis, as exemplified by their effectson the motility of SUM cells derived from human inflammatory breastcancer (IBC) cells and RhoC-overexpressed human mammary epithelium(HME-RhoC) cells as described herein in the Examples. Without beingbound by theory, it is believed that the thiopurine-mediated inhibitionof cell motility occurs because the thiopurine targets and inhibitsRhoC. A molecular mechanism for the thiopurine mediated RhoC inhibitionalso has been shown in which 6-TPs are converted in cells into the6-thioguanosine phosphate (6-TGNP), which in turn reacts with the Cys²⁰side chain of the RhoC GXXXXGK(S/T)C (SEQ ID NO:1) motif to produce a6-TGNP-RhoC disulfide adduct. A redox agent enhances this disulfideformation process. The adduct formed impedes RhoC guanine nucleotideexchange, which populates an inactive RhoC. This result is in contrastto the targeting of Ras by 6-TG-redox agent combinations, which wasminimal because the redox-active Ras cysteine is not located at itsnucleotide-binding site. In some embodiments, formation of the Rhoprotein-disulfide adduct occurs in GTPases that possess theGXXXXGK(S/T)C (SEQ ID NO:1) motif such as RhoC, RhoA, and Rac. RhoCtherefore is a target for treatment of diseases related to RhoC by usinga combination of redox-sensitive purine compounds and a redox agent asan effective chemotherapeutic agent to inhibit or terminate themetastasis of IBC. In some embodiments of treating cancer metastasis,for example, by inhibiting RhoC, the redox-sensitive purine administeredis 8-thioguanine (8-TG) and the redox agent is the nitric oxide (NO)donor DETA/NO.

In some embodiments, the disease or condition to be treated is prostatecancer. In some embodiments, the Rho family GTPase that is inhibited isa Rac protein. In some embodiments, high-grade prostatic intraepitheliumneoplasia (HG-PIN) is treated. HG-PIN, like prostate cancer, isassociated with overexpression of Rac proteins.

In some embodiments, the disease or condition to be treated is a bloodvessel disease. In some embodiment, the Rho family GTPase that isinhibited is RhoA. In some embodiments, the blood vessel disease ishypertension, and the methods of the invention promote vasorelaxation ofblood vessels, and improvement of the condition.

In some embodiments, the disease or condition to be treated is immunerelated. In some embodiments, the disease or condition involves T-cells,which includes, but is not limited to, cell-mediated hypersensitivity,such as delayed type hypersensitivity and T-cell-mediated cytotoxicity,and transplant rejection; autoimmune diseases, such as systemic lupuserythematosus, Sjogren syndrome, systemic sclerosis,inflammatory-myopathies, mixed connective tissue disease, andpolyarteritis nodosa and other vasculitides, inflammatory bowel disease,ulcerative colitis and rheumatoid arthritis. In some embodiments, theRho family GTPase that is inhibited is Rac1.

The redox-sensitive purine that can be administered to treat thediseases or conditions described herein is not limiting and includes allof the compounds as described herein. In some embodiments, theredox-sensitive purine is a 6-thiopurine, such as 6-thioguanine. In someembodiments, the redox-sensitive purine is a 8-thiopurine, such as8-thioguanine. In some embodiments, the redox-sensitive purine is acompound of formula (I).

In some embodiments, the invention further provides methods of screeningfor compounds that inhibit a redox-sensitive GTPase protein, including aRho or Rab family GTPase protein. In some embodiments, the methodscomprise contacting a composition comprising a Rho or Rab family GTPasewith a redox-sensitive compound, optionally in the presence of a redoxagent, wherein the Rho or Rab family GTPase and the redox-sensitivecompound form an adduct. In some embodiments, the method furthercomprises detecting the adduct and/or assaying for the activity of theRho or Rab family GTPase. In some embodiments, the compound is aredox-sensitive purine as described herein. In some embodiments, thecompound is not a purine compound, but is redox-sensitive and is capableof forming an inhibitory adduct with a Rho or Rab family GTPase.

The modulation or inhibition of Rho or Rab family GTPases can bemeasured according to any assay typically used to measure Rho or RabGTPase activity. In some embodiments, the activity can be measured byanalyzing GTP hydrolysis, binding to Rho-GEF or Rab-GEF etc. Variousassay methods for monitoring the activation state of a Rho GTPase, forexample, are known and can be incorporated into the screening methods ofthe invention. One assay, the Rho effector pull-down assay, wasoriginally developed for RhoA GTPases (Ren et al. EMBO J. 18: 578-585(1999)) and for Rac1/Cdc42 GTPases (Benard et al. J. Biol. Chem. 274:13198-13204 (1999)) and is a classical and widely used assay. The methodinvolves capture of activated Rho GTPase proteins by effectors bound tobeads, release of the GTPase protein from the beads, separation of thebeads from the released GTPase protein, followed by SDS-PAGE andanalysis of the GTPase protein by western blotting. There are severalcell-based assays that use fluorescent bio-probes to detect activatedRho GTPases (Pertz et al. J. Cell Sci. 117: 1313-1318 (2004)). Severalversions of this type of assay rely on a reporter system to monitor invivo Rho GTPase activation. These cell-based assays, therefore, do notmonitor the actual endogenous levels of the GTPase (Itoh et al. Mol.Cell Biol. 22: 6582-6591 (2002); Pertz et al. Nature 440: 1069-1072(2006); Vadim et al. Science 290: 333-337 (2000)). Other versions ofcell-based assays use effector domains linked directly to anenvironmental dye to monitor endogenous in vivo GTPase activation. Anenzymatic based method to detect Rho activation has been described (Chenet al. J. Biol. Chem. 278: 2807 (2003)). The assay utilizesGST-effector-GBD to affinity precipitate active GTP-Rho. GTP is elutedand converted to ATP in a coupled enzymatic assay. ATP is then measuredby the firefly luciferase method.

In some embodiments, downstream or biological effects of Rho or Rabproteins can be analyzed to screen for inhibitory Rho or Rab GTPasecompounds. In some embodiments, cell invasiveness is assayed as adownstream effect of Rho GTPase proteins (See FIG. 14 and the Example 3,herein). In some embodiments, changes in the cytoskeleton, or vesiculartrafficking can be detected in the presence and the absence of theredox-sensitive compound, as an indirect means of detecting Rho or RabGTPase activity. An automated cell-based Rho activation assay has alsobeen described (Teusch et al., 2006, Assay and Drug Devel., 4: 133)based on the ability of Rho to regulate the actin cytoskeleton. A RhoGTPase activation assay that measures cytoskeletal changes is alsodiscussed in U.S. Application Pub. No.: 20060177816. The disclosures ofthe above cited documents which pertain to methods for assaying of RhoGTPase activity are incorporated by reference herein.

Various cell types can be screened for Rho or Rab GTPase inhibition inaccordance with the above described methods, including cancer cells,such as breast cancer cells, immune cells, such as T-cells, and smoothmuscle cells. In addition, specific Rho or Rab GTPases can be screenedfor inhibition. In some embodiments, Rho GTPase inhibitors can beidentified that are more selective for a particular Rho GTPase familymember over other family members. The identification of Rho or Rabselective inhibitors will likely improve any toxicity that might beassociated with use of inhibitors that target all Rho or Rab familyGTPases.

The examples disclosed below illustrate embodiments of the invention andare not intended to limit the scope. It is evident to those skilled inthe art that modifications or variations can be made to the embodimentsdescribed herein without departing from the teachings of the presentinvention.

EXAMPLES Example 1 Small GTPase-Targeting Action of 6-Thioguanine and8-Thioguanine

This example provides studies on the investigations of the Rho and Rascellular targeting action of 6-thioguanine and 8-thioguanine with aredox agent.

Actions of 6-thioguanine on Rho.

RhoC-overexpressed mammary epithelium (HME-RhoC) cells were used toexamine the effect of 6-TG and/or a redox agent on RhoC. When HME-RhoCcells were treated with 6-TG (1 μM), nearly 80% of the cells underwentapoptosis within a day. A decrease in the cell population of HME-RhoCcells also was observed when other 6-TG analogs such as 6-MP and AZAwere used (not shown). This result is consistent with previousobservations that 6-TG and its analogs induce cell death for certaintumor cell lines via altering the stability of DNA (Lage et al. J.Cancer Res. Clin. Oncol. 125, 156-165 (1999); Yan et al. Clin. CancerRes. 9, 2327-2334 (2003); Kaba et al. J. Clin. Oncol. 15, 1063-1070(1997); Bae et al. Cancer Lett. 126, 97-104 (1998); Matheson et al. Adv.Exp. Med. Biol. 457, 579-583 (1999); Liu et al. Leukemia 16, 223-232(2002); Morgan et al. Cancer Res. 54, 5387-5393 (1994)). Some cells(20%-25% of the initial cells), however, persistently resistedadditional treatments with 6-TG and even slowly regrew in the presenceof 6-TG or its analogs such as 6-MP and AZA. It is unclear if theresistance of these cells to 6-TG or its analogs is directly induced bytreatment with 6-TG or its analogs. However, this resistance could be aproblem for patients over the course of long-term treatment with 6-TG orits analogs (de Boer et al. Nat. Clin. Pract. Gastroenterol. Hepatol. 4,686-694 (2007); Karran et al. Nat. Rev. Cancer 8, 24-36 (2008); Cuffariet al. Can. J. Physiol. Pharmacol. 74, 580-585 (1996)).

When the 6-TG-resistant HME-RhoC cells were further treated with an NOdonor (diethylenetriamine/NO, DETA/NO or S-nitrosoglutathione, GSNO; 10μM), the invasiveness of the 6-TG-resistant HME-RhoC cells drasticallydeclined (retained less than 5% of their original invasiveness; FIG. 5).Because the half-life of redox agents, GSNO and DETA/NO, is ˜8 and ˜20h, respectively, at pH 7.4 and 37° C. (Maragos et al. J. Med. Chem. 34,3242-3247 (1991); Mooradian et al. J. Cardiovasc. Pharmacol. 25, 674-678(1995); Floryszak-Wieczorek et al. Planta 224, 1363-1372 (2006)), theirtreatment intervals were 8 and 20 h to ensure the concentration of NOremained for the duration of the experiment at a minimal 50% of theinitial concentration of NO in the culture media.

To determine if the loss of invasive cell mobility associated with RhoCwas attributable solely to either 6-TG (or its analogs) or to thecombined effect of 6-TG with a redox agent, RhoC in a complex with6-TGNP was probed with RhoC antibody, digested with trypsin, andanalyzed with Matrix-Assisted Laser Desorption/Ionization time-of-flight(MALDI-TOF) (FIG. 6). An unusual mass peak, 1475.6 Da., was detectedthat is assigned as an unusual peptide-6-TGDP adduct; TC²⁰LLIVFSK-6-TGDP(FIG. 6). Supporting the experiment, the peptide-6-TGDP adduct also wasdetected in the in vitro sample of the RhoC 6-TGNP complex digested withtrypsin, but not from the RhoC C20S mutant in the complex with 6-TGDP(not shown). The results suggest that the 6-TGNP, derived from 6-TG (orits analogs), binds to RhoC and/or covalently links to the redox-activeRhoC Cys²⁰ side chain.

In view of structural considerations (Ihara et al. J. Biol. Chem. 273,9656-9666 (1998); Dias et al. Biochemistry 46, 6547-6558 (2007))associated with a previous study (Heo et al. Biochemistry 45,14481-14489 (2006)) and the detection of the peptide-6-TGTP chimericadduct (FIG. 6), a molecular mechanism is proposed that accounts for the6-TGNP-mediated RhoC inactivation which leads to loss of invasive cellmobility (FIG. 7): (i) 6-TGTP, derived from 6-TG, binds to the RhoC GTPbinding site, (ii) a redox agent (i.e., .NO₂) reacts with the RhoC Cys²⁰side chain to produce a RhoC-Cys²⁰ thiyl radical. This thiyl radicalthen further reacts with the thiolate moiety of the bound 6-TGNP toproduce a RhoC disulfide anionic radical (6-TGNP-RhoC Cys²⁰{tilde over(.)}⁻). The protein disulfide anionic radical can be quenched by acellular molecule (i.e., glutathione or ascorbic acid) to produce a6-TGNP-RhoC Cys²⁰; (iii) although the γ phosphate moiety of thechemically linked 6-TGTP can be hydrolyzed into 6-TGDP by Rho GAPs, itcannot be released from the RhoC by the action of a Rho GEF (e.g., Dbs)because of its disulfide linkage to RhoC. This linkage halts the Rho GEFDbs-mediated GTP/GDP cycle, thereby terminating RhoC-dependent cellmotility. The mechanism provides an explanation for the 6-TG-induceddecline of cell invasiveness associated with RhoC in the presence of aredox agent.

The combined actions of 6-TG and a redox agent have been shown to bevery effective for cellular RhoC inactivation (FIG. 5). Notably, acertain type of IBC is caused by the overexpression of RhoC (van Golenet al. Clin. Cancer. Res. 5, 2511-2519 (1999); Clark et al. Nature 406,532-535 (2000). Hence, by mass action, a sequential treatment with 6-TGand a redox agent can target cellularly populated RhoC to inactivateRhoC, which ends invasive cell mobility and terminates the propagationof invasive tumors.

Although the molecular mechanism shown in FIG. 7 explains the action of6-TGNP that targets one of the Rho GTPases, RhoC, the mechanism also isapplicable to other GTPases having a redox-active cysteine in theirnucleotide-binding GXXXXGK(S/T)C (SEQ ID NO:1) motif. For example,because Rac1 has the GXXXXGK(S/T)C motif, 6-TGNP derived from 6-TG isexpected to target Rac1 to produce a 6-TGNP-Rac1 disulfide adduct.Recent in vitro kinetic results show that in the presence of a redoxagent, 6-TGNP reacts with Rac1 Cys¹⁸ (equivalent to RhoC Cys²⁰) toproduce a 6-TGNP-Rac1 disulfide adduct (not shown). Moreover, otherstudies conducted without adding a redox agent suggest that 6-TGNP,coupled with the action of Vav, can specifically target Rac1, and resultin relatively more inactivation of Rac1 than of other Rho proteins(Tiede et al. J. Clin. Invest. 111, 1133-1145 (2003); Poppe et al. J.Immunol. 176, 640-651 (2006)). However, as stated, these studies did notconsider the action of a redox agent on the 6-TGNP-mediated inactivationof Rho GTPases.

Actions of 6-thioguanine on Ras.

Human bladder carcinoma (T24) and fibrosarcoma (HT1080) cells owe theircancerous properties, respectively, to the constitutively activatedH-Ras G12V and N-Ras Q61K (Reddy et al. Nature 300, 149-152 (1982);Brown et al. EMBO J. 3, 1321-1326 (1984)); these T24 and HT1080 cellswere used to examine the effect of 6-TG and/or a redox agent on Ras.Cell viability was examined employing3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Cabello et al. Int. J. Oncol. 23, 697-704 (2003)).

As with HME-RhoC cells, when T24 or HT1080 cells were treated with 6-TG(1 μM), most cells underwent apoptosis, and thus the cell populationdrastically declined within a day (not shown). Similar to the situationwith HME-RhoC, when cells were treated with 6-TG, ˜20% of the initialcells resisted additional treatment with 6-TG and slowly regrew in thepresence of 6-TG (not shown). When 6-TG-resisting T24 or HT1080 cellswere continuously treated with both 6-TG (1 μM; every 48 h) and DETA/NOor GSNO (10 μM for every 20 and 8 h, respectively) for longer than ˜10days, a decrease in the viability of 6-TG-resisting cells was observed.The decrease in viability of T24 (or HT1080) cells was likely caused bythe inactivation of Ras, because a significant amount of Ras (per cellmass) was inactivated by the 6-TG treatment (1 μM; every 48 h) andDETA/NO (10 μM; every 20 h) for longer than 10 days (FIG. 8).

An Electrospray Mass Spectrometry (ESI-MS) analysis shows that adetectable amount of 6-TGNP bound to Ras in T24 or HT1080 cells wasobserved after ˜8-10 days from the initial treatment with 6-TG (FIG. 9).In the presence of both 6-TG (1 μM; every 48 h) and DETA/NO (2 μM; every20 h) or GSNO (10 μM; every 8 h), 6-TGNP was barely detectable, but a463.3 Dalton peak, which is assigned to 5-guanidino-4-nitroimidazolediphosphate (NIm-DP), was clearly present (FIG. 9). NIm-DP was probedonly with inactivated Ras, suggesting that formation of NIm-DP coupleswith Ras inactivation in these cells (FIG. 8). Notably, NIm-DP isidentified as a product of the degradation of the GDP-NO₂ adduct, whichis released from Ras upon treatment with .NO₂ (Heo et al. J. Mol. Biol.346, 1423-1440 (2005)).

Although T24 or HT1080 cells produce Ras bound NIm-DP in the presence of6-TG and a redox agent, a 6-TGNP-Ras disulfide adduct was not detectablein these tumor cells. It is believed that this is because, unlike withthe GXXXXGK(S/T)C (SEQ ID NO:1) motif-containing Rho proteins such asRhoC, Rac 1, and Cdc42, the 6-TGNP target site of the NKCD (SEQ ID NO:3)motif-containing Ras GTPases is remote from the Ras nucleotide-bindingsite (See FIGS. 2 and 3) (Nassar et al. Nature 375, 554-560 (1995);Dumas et al. Structure 7, 413-423 (1999); Pai et al. Nature 341, 209-214(1989); Brunger et al. Proc. Natl. Acad. Sci. U.S.A. 87, 4849-4853(1990); Kraulis et al. Biochemistry 33, 3515-3531 (1994); Tong et al. J.Mol. Biol. 217, 503-516 (1991); Ito et al. Biochemistry 36, 9109-9119(1997); Scheidig et al. Structure 7, 1311-1324 (1999)).

Given that .NO₂ can oxidize the sulfur atom of thiolate to produce athiyl radical, .NO₂ may react directly with the sulfur atom of theRas-bound 6-TGNP to produce a 6-TGNP-thiyl radical (FIG. 10). .NO₂ canalso target the redox-sensitive Ras Cys¹¹⁸ side chain if the sulfur siteis available. The Ras Cys¹¹⁸ side chain thiyl radical that is formed canproduce the 6-TGNP-thiyl radical via the Phe²⁸ side chain as suggestedin a previous study (Heo et al. J. Mol. Biol. 346, 1423-1440 (2005)).Importantly, the 6-TGNP-thiyl radical is equivalent to the oxygenradical in the radical-based Ras GNP dissociation mechanism (FIG. 10).Hence, the 6-TGNP-thiyl radical likely proceeds to perturb theRas-binding interactions with 6-TGNP and then reacts with another .NO₂,resulting in the release of chemically modified 6-TGNP, 6-TGNP-NO₂ (FIG.10). If so, the unstable 6-TGNP-NO₂ is degraded into NIm-DP by releaseof a carbon dioxide sulfide (O═C═S) (FIG. 10). As shown in FIG. 10, themechanism of NIm-DP formation from the 6-TGNP-NO₂ adduct is likelysimilar to the decarboxylation of GNP-NO₂ to produce NIm-DP (Heo et al.J. Mol. Biol. 346, 1423-1440 (2005)).

In accounting for the proposed mechanism (FIG. 10), the equilibriumbetween the cellular 6-TGNP and the 6-TGNP-bound Ras also can beenhanced by .NO₂. However, how .NO₂ inactivates the 6-TGNP-bound Ras isless clear. Enhancement of the equilibrium between the cellularly free6-TGNP and the oncogenic Ras-bound 6-TGNP cannot serve to inactivate Rasunless the cellular ratio of 6-TGTP/6-TGDP is <1. Another possibility isthat a redox agent (i.e., .NO₂) and Ras downstream effector proteinscompete for the 6-TGTP-bound Ras. Therefore, somehow the presence of aredox agent serves to reduce some amount of the mechanically stablestate of oncogenic Ras that is bound with 6-TGTP and is capable ofinteracting with its downstream effector proteins.

Cytotoxic effects associated with chronic exposure to 6-TG and itsanalog drugs are all but inevitable. The 6-TG cytotoxicities associatedwith the DNA-targeting 6-TdGNP are well-known (Karran, P. Br. Med. Bull.79-80, 153-170 (2006); O'Donovan et al. Science 309, 1871-1874 (2005);Hsieh et al. Mech. Ageing Dev. 129, 391-407 (2008); Casorelli et al.Anticancer Agents Med. Chem. 8, 368-380 (2008); Daehn et al. Cancer Res.69, 2393-2399 (2009)). This study is the first to demonstrate that anaccumulation of 6-TGNP over the course of long-term treatment with 6-TGcan interfere, in the presence of a redox agent, with cellular proteinRas GTPases. Given that Ras plays a key role in cell signaling events,deregulation and/or misregulation of Ras is detrimental and thuscytotoxic to cells.

Small GTPase-Targeting Action of 8-Thioguanine.

To develop a better agent that targets the GXXXXGK(S/T)C (SEQ ID NO:1)motif of Rho proteins, we have screened the effects of variousthiopurine analogs on the invasive cell mobility of HME-RhoC cells. Thefollowing is a list of those analogs screened: 8-thioguanine(2-amino-8-mercapto-1H-purin-6(9H)-one), 8-methylthioguanine(2-amino-8-(mercaptomethyl)-1H-purin-6(9H)-one), 7-methylthioguanine(2-amino-7-(mercaptomethyl)-8,9-dihydro-1H-purin-6(H)-one), and7-thioguanine (2-amino-7-mercapto-8,9-dihydro-1H-purin-6(H)-one). [7-and 8-thioguanines are commercially available. Other analogs wereobtained from Azopharma Drug Development Services, Miramar, Fla.] Ofthese several thiopurine analogs screened, 8-thioguanine (8-TG) mosteffectively inhibits the invasive cell mobility of HME-RhoC cells in thepresence of a NO donor DETA/NO (see details below).

1. RhoC and 8-thioguanine.

On the basis of screening results associated with the inhibitory actionof 8-TG on the invasive cell mobility of HME-RhoC cells, the detailedcellular effect of 8-TG on HME-RhoC cells in the presence and absence ofa redox agent was further analyzed.

(a) Effects of 8-TG and/or a Redox Agent on HME-RhoC Cells.

The invasive cell mobility of HME-RhoC cells declined (˜10%) within aday after treatment with only 8-TG (1 μM) (FIG. 11). The invasive cellmobility of HME-RhoC cells further declined (˜60%) over the course of 5days of continuous treatment with only 8-TG (1 μM) (FIG. 11). However,unlike with 6-TG, the apoptotic effect of 8-TG on HME-RhoC cells wasminimal because less than 5% of HME-RhoC cells treated with 8-TG (1 μM)underwent apoptosis within a day. A delayed minor apoptosis (˜5%) wasobserved in HME-RhoC cells over the course of a week of treatment with8-TG (1 μM).

When HME-RhoC cells were treated with 8-TG for a week, followed bytreatment with an NO donor (DETA/NO or GSNO; 10 μM for every 20 or 8 h,respectively), virtually all of the invasive mobility of HME-RhoC cellswas immediately terminated. Intriguingly, despite termination of cellinvasiveness, the viability of the HME-RhoC cells treated with an8-TG-redox agent was almost the same as that of the untreated HME-RhoCcells.

(b) Potential Mechanistic Action of 8-TG and a Redox Agent on theTermination of Cell Mobility of HME-RhoC Cells.

The structural feature of the nucleotide-binding pocket of RhoC (PDB1GCO) in conjunction with the proposed molecular mechanism for RhoC(FIG. 7) explains the inhibitory action of 8-TG on the invasive cellmobility of HME-RhoC cells, where the distance between the sulfur atomof the RhoC Cys²⁰ side chain and the sulfur atom at the C₈ position ofbase (˜2.1 Å; c.f., a direct distance between the RhoC Cys²⁰ sulfur atomand the C₈ carbon is ˜3.6 Å) is shorter than that of the sulfur atom ofthe RhoC Cys²⁰ side chain and the sulfur atom at the C₆ position of base(˜6.9 Å) (FIG. 12). The putative distance between the sulfur atom of theRhoC Cys²⁰ side chain and the sulfur atom at the C₈ position of the base(˜2.1 Å) is optimal for a disulfide bond distance. In addition, insilico analysis using MM2 and MOPAC energy minimizations performed by 3DChem (Cambridge software) suggests that the disulfide bonding torsionconstraint between the sulfur atom of the RhoC Cys²⁰ side chain and thesulfur atom at the C₈ position of the base is less than the disulfidebonding torsion constraint between the sulfur atom of the RhoC Cys²⁰side chain and the sulfur atom at the C₆ position of 6-TG base.

(c) Implication of the Action of 8-TG and a Redox Agent on CellsOverexpressing RhoC.

These results indicate that the effect of 8-TG in conjunction with aredox agent for the termination of the RhoC-mediated cell invasivenessof HME-RhoC cells was much more effective than a 6-TG-redox combinationin terminating the RhoC-mediated cell invasiveness of HME-RhoC cells.

2. Ras and 8-thioguanine.

The cancerous property of T24 and HT1080 cells is because of thepresence of constitutively active Ras (Li et al. Br. J. Cancer. 92,80-88 (2005); Rait et al. Bioconjug. Chem. 11, 153-160 (2000); Chen etal. J. Biol. Chem. 271, 28259-28265 (1996); Liu et al. Anticancer Res.17, 1107-1114 (1997); Plattner et al. Proc. Natl. Acad. Sci. U.S.A. 93,6665-6670 (1996); Gupta et al. Mol. Cell. Biol. 21, 5846-5856 (2001)).Hence, as with 6-TG (FIGS. 8 and 9), if 8-TG targets and perturbs RasGTPase, the cancerous growth of these tumor cells will be altered.However, 8-TG (1 μM) in combination with an NO donor (DETA/NO or GSNO;10 μM for every 20 or 8 h, respectively) did not affect viability andanchorage-independent growth of T24 and HT1080 cells (not shown). Theexpression and activity of Ras in these cells also were virtuallyunchanged (not shown). These results indicate that, unlike with 6-TG,the cytotoxic effect of 8-TG with a redox agent associated with Ras isminimal. Therefore, 8-TG together with a redox agent may not be able toperturb Ras activity in these cells.

Example 2 Examination of the Biochemical Effects of 6-TGNP or 8-TGNP onRhoC GTPase in the Presence and Absence of a Redox Agent and/or Dbs

Kinetic Assays on the Role of a Redox Agent in the Formation of aDisulfide Bond Between 6- or 8-TGNP and a RhoC Protein:

To explore the effect and molecular mechanism of the formation of the 6-or 8-TGNP-RhoC adduct via the formation of a 6- or 8-TGNP RhoC disulfideon the RhoC GNE, kinetic analyses were performed.

A kinetic assay method using radiolabeled GDP ([³H]GDP) was established.The apparent dissociation constants (^(app)K_(D 6-TGDP)) of Rho GTPases(including RhoA, Cdc42, and Rac1/2/3) and Ras with 6-TGDP weredetermined (FIG. 13). The results of this preliminary kinetic studyprovided strong support for the mechanism proposed for the role of6-TGNP in RhoC inactivation (FIG. 7).

Studies show that binding interactions between Rho GTPases and 6-TGNPwere weaker than binding interactions between Ras and 6-TGNP (Tiede etal. J. Clin. Invest. 111, 1133-1145 (2003); Poppe et al. J. Immunol.176, 640-651 (2006)) although an ^(app)K_(D 6-TGDP) value of RhoC wasnot determined in these studies. FIG. 13 shows that the^(app)K_(D 6-TGDP) of RhoC [³H]GDP with 6-TGDP is similar to that ofRhoA but much smaller than that of Ras. The true dissociation constant(^(true)K_(D 6-TGDP)) of Ras, RhoC, RhoA and Rac1 for 6-TGDP was thencalculated to be ˜20.7 pM, 212 μM, 208 μM, and 158 μM [6-TGDP] by usinga compensation equation,^(true)K_(D 6-TGDP)=^(app)K_(D 6-TGDP)/(1+[GDP]/^(true)K_(D GDP)) (95),in conjunction with the values given [(^(app)K_(D 6-TGDP) of Ras, RhoC,RhoA, and Rac1=2300, 230, 224, and 245 μM (FIG. 13) and^(true)K_(D GDP)=9 pM, 12 μM, 13 μM and 1.8 μM, respectively. The^(true)K_(D GDP) of Ras, RhoA and Rac1 was determined previously (Lenzenet al. Biochemistry 37, 7420-7430 (1998); Heo et al. Biochemistry 44,6573-6585 (2005)) and the ^(true)K_(D GDP) of RhoC was furtherdetermined. Example: ^(true)K_(D 6-TGDP) for Ras=2300 μM/(1+1 μM GDP/˜9μM)=−20.7 μM [6-TGDP]]. The results suggest that the binding interactionof Rho GTPases with 6-TGDP is ˜15-fold weaker than with GDP.

FIG. 13B shows that the ^(app)K_(D) of RhoC 6-TGDP with [³H]GDP in theabsence of DTT was much larger than that of RhoC with GDP in thepresence DTT. The ^(app)K_(D [3H]GDP) was converted into^(true)K_(D [3H]GDP) by using a compensation equation,^(true)K_(D [3H]GDP)=^(app)K_(D [3H]GDP)/(1+[6-TGDP]/^(true)K_(D GDP))(Segel, I. H. (1993) Enzyme kinetics, A Wiley-Interscience publication,New York) (see above)). The calculated ^(true)K_(D [3H]GDP) values ofRhoC in the presence and absence of DTT were estimated to be 516 μM and923 μM, respectively. However, the ^(app)K_(D [3H]GDP) values of Ras inthe presence and absence of DTT were not significantly changed when theywere converted into ^(true)K_(D [3H]GDP) values (^(true)K_(D [3H]GDP)values of Ras=4.7 and 4.8 nM, respectively). Unlike with Ras, such alarge ^(true)K_(D [3H]GDP) value of RhoC estimated in FIG. 13B (923 μM)in the absence of DTT suggests that [³H]GDP cannot compete with the RhoCbound 6-TGDP. The smaller ^(true)K_(D [3H]GDP) value of RhoC in thepresence of DTT suggests that [³H]GDP was able to drive out 6-TGNP fromRhoC in the presence of DTT. One possible explanation for these resultsis that the chemical modification of RhoC with 6-TGDP to produce the6-TGNP-RhoC adduct blocks release of 6-TGDP from RhoC (see FIG. 7).Treatment with DTT, a reducing agent, disrupts the disulfide bondbetween RhoC and 6-TGDP, liberating 6-TGDP from RhoC, and therebypermitting [³H]GDP to bind to RhoC.

Example 3 Insight into the Termination of the Invasive Motility of TumorCells Derived from Inflammatory Breast Cancer by 6-Thiopurines

Cell Culture and Treatments.

SUM cells were cultured according to the cell culture protocol providedby the vendor (Asterand, Detroit Mich.). Transfection of human mammaryepithelial cells with wild type RhoC and mutant C20S RhoC to produceHME-RhoC and HME-C20S RhoC, respectively, as well as culturing of theseHME-RhoC and HME-C20S RhoC cells were performed according to knownprocedures (van Golen et al., Mol. Cancer Ther. 1, 575-583 (2002)).Human colon adenocarcinoma (cell-line, SW480) and hepatocellularcarcinoma (cell-line, HCCLM3) cells were cultured according to the cellculture protocol provided by the vendor (American Type CultureCollection, Manassas, Va.). Cells were treated with 6-TG (1 μM) and/or aNO-releasing agent diethylenetriamine/nitric oxide (DETA/NO) (10 μM) orS-nitrosoglutathione (GSNO) (10 μM) at every ˜24 and/or ˜20 or ˜8 h,respectively, for three days to maintain a minimal 50% of the initialconcentration of NO in the culture media (Heo et al., Biochemistry 49,3965-3976 (2010)). The treatment time interval for 6-TG is based onempirical reasoning that three consecutive treatments with 6-TG for upto four days will not result in an overdose of 6-TG, but will ensurethat the level of 6-TG is higher than 1 μM in the culture media. Theestablished cell treatment time interval for DETA/NO or GSNO is becausethe half-life of DETA/NO and GSNO is ˜20 and ˜8 h, respectively, at pH7.4 and 37° C. (Maragos et al. J. Med. Chem. 34, 3242-3247 (1991);Mooradian et al. J. Cardiovasc. Pharmacol. 25, 674-678 (1995); andFloryszak-Wieczorek et al. Planta 224, 1363-1372 (2006)).

Cell Motility Assay.

An invasion assay using Matrigel (BD Biosciences, Bedford Mass.),performed as described previously except for minor modification (vanGolen et al. Clin. Exp. Metastasis 14, 95-106 (1996)), was the majormethod used in examining the effect of 6-TG and/or a redox agent onSUM149, HME-RhoC, or HME-C20S RhoC cells. The top chamber of a Transwellfilter (6.5 mm with 8 μm pores, Costar; Corning, N.Y.) was coated with a10 μL aliquot of 10 mg/mL Matrigel, and the lower chamber of theTranswell was filled with either serum-free or serum-containing media.Sample cells were prepared to resuspend cells in a serum-free mediumwith 0.1% BSA at a concentration of 4×10⁵ cells/mL. The sample cells(0.5 mL) were added to the top chamber of a Transwell filter, overlaidwith a solution (0.1 mL) containing 6-TG and/or DETA/NO or GSNO, andthen incubated for three days at 37° C. in a 10% CO₂ incubator. Whennecessary, the top chamber was repeatedly overlaid with the solution(0.1 mL) containing 6-TG and/or DETA/NO or GSNO as indicated above. TheTranswell filters were fixed with methanol, stained with haematoxylinand eosin, and cells in the serum-containing samples were counted. Thenumber of cells that had invaded the serum-free, medium-containing lowerchambers was used as a control background.

The Colorimetric-based QCM Cell Invasion Assay kit (Millipore,Billerica, Mass.) was used for the time-dependent cell migration assayof SUM149, SW480, and HCCLM3 cells. Serum-free sample cells (2×10⁵cells/mL) were prepared as indicated in the section of the matrigelinvasion assay. The serum-free sample cells (0.25 mL) were loaded to therehydrated upper chamber. The cell-containing upper chamber was thenoverlaid with a solution (0.05 mL) containing 6-TG and/or DETA/NO orGSNO. Serum-free or serum-containing media (0.5 mL) was added to thelower chamber. The plate covered was then incubated for one to four daysat 37° C. in a 10% CO₂ incubator. Like with the matrigel invasion assay,the upper chamber was repeatedly overlaid with the solution (0.05 mL)containing 6-TG and/or DETA/NO to maintain the desired level of 6-TG andNO in the upper chamber. The fraction of cells migrated from the upperchamber to the lower chamber was then measured colorimetricallyaccording to the procedure provided by the manufacture.

Clonogenic, Viability, and Apoptotic Assays.

When necessary, various other assays such as a soft agar-basedclonogenic assay (Yamashita et al. Oncogene 18, 4777-4787 (1999); Ghataket al. J. Biol. Chem. 277, 38013-38020 (2002)), a cell viability assayemploying 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(Vistica et al. Cancer Res. 51, 2515-2520 (1991); Sato et al. CancerLetters 112, 181-189 (1997)), and an apoptosis assay using the ApoAlertCaspase-3/8 Colorimetric Assay Kit (Takara, Japan) also were performedto examine the effect of a NO donor and/or 6-TG on HME-RhoC or HME-C20SRhoC cells.

RhoC Activity Assay in Cells.

Cells were lysed in an extraction buffer containing 50 mM NaCl, 5 mMMgCl₂, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mMdiethylenetriaminepentaacetic acid (DTPA), and 0.1% NP40 in 150 mMTrisHCl (pH 8.0). The content of active RhoC in cells then wasdetermined by using Western analysis that relies on a monoclonalanti-RhoC antibody (Biocompare, South San Francisco, Calif.). Todetermine RhoC activity, the colorimetric RhoA activation assay BiochemKit (Cytoskeleton, Denver, Colo.) was used essentially as described inthe previous study (Hall et al. Neoplasia 10, 797-803 (2008)) exceptthat the RhoC specific antibody (vide supra), instead of the RhoAantibody included in the assay kit, was used for this analysis.

Mass Spectrometric Analysis.

Cell lysates in an extraction buffer were incubated with a monoclonalanti-RhoC antibody precoupled to glutathione-agarose beads. The beadswere collected by centrifugation and washed three times with theextraction buffer. RhoC proteins on beads were then eluted with asolution containing 50% methanol, 0.1% formic acid, 0.1 mM MgCl₂ 1 mMEDTA, and 0.1 mM DTPA. The protein portion and RhoC-released nucleotidewere separated by brief centrifugation (3,000×g for 5 min). The proteinportion from the immunoprecipitated RhoC was resuspended in a buffercontaining 1 mM EDTA and 0.1 mM DTPA in 50 mM TrisHCl (pH 7.4), thendigested with trypsin for 10 h, and analyzed with electrospray massspectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) in thepositive ion mode ([molecular mass+H]⁺).

Kinetic Assays.

Transition metal-free assay buffer and vials as described in a previousstudy (Heo et al. Biochemistry 49, 3965-3976 (2010)) were used for allkinetic assays. The kinetic assay buffer contains the highest grade of50 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, and 0.1 mM DTPA in 50 mM TrisHCl (pH7.4). Before performance of any of the assays, all protein samples alsowere dialyzed with a metal-free buffer under anaerobic conditions. Aswith RhoA and Rac1 (Heo et al. J. Biol. Chem. 280, 31003-31010 (2005)),C20S RhoC were expressed in E. coli and purified using anionic and sizeexclusion columns.

(a) A competitive binding assay was performed to determine the bindingaffinity of Rho GTPase and 6-TGDP. The radiolabeled [³H]GDP-loaded Rhoprotein (1 μM) was titrated with various concentrations of 6-TGDP. Thetitrated Rho protein sample then was spotted onto a nitrocellulosemembrane. Each membrane was washed three times with the assay buffer,and radioactivity was determined using a Beckman-Coulter scintillationcounter. The radioactivity of the sample (Rho protein-bound [³H]GDP) wasconverted into, and thus plotted, as the fraction of mol GDP per moltotal Rho protein. The apparent dissociation constant (^(app)K_(D)) ofthe Rho protein with [³H]GDP in the presence of 6-TGDP was determined byusing Prism software to fit the titration curve to a hyperbola.

(b) The effect of redox agents such as NO, .NO₂, and N₂O₃ on the bindinginteraction between RhoC proteins and GDP or 6-TGDP was examined underN₂-filled serum-stoppered anaerobic experimental conditions (O₂<3 ppm).These anaerobic experimental conditions were necessary to block thereaction of NO or .NO₂ with O₂ to produce higher oxides. The contents ofNO and .NO₂, respectively, in the sealed assay vials were determined byusing a hemoglobin assay and NO₂ ⁻/NO₃ ⁻ Assay Kit-C II (Dojindo) underanaerobic conditions (Heo et al. J. Biol. Chem. 280, 31003-31010 (2005);Heo et al. Biochemistry 43, 2314-2322 (2004)). N₂O₃ was generated bymixing NO and .NO₂ 1:1 stoichiometrically. However, because the N₂O₃thus formed can be decomposed into NO and .NO₂, the content of N₂O₃ neednot be at 100%. To examine the redox property of RhoC with GDP, the[³H]GDP-loaded RhoC protein (1 μM) was treated with redox agents (˜3 μM)in the presence of free GDP (10 μM). The fraction of the redoxagent-mediated release of the bound [³H]GDP per Rho protein over timewas determined as noted above, and the result was plotted against time.The rates of RhoC GDP dissociation in the presence of redox agents weredetermined by using Prism software to fit the result to a one-phaseexponential decay.

(c) To test the Rho 6-TGDP-binding interaction in the presence of redoxagents, the 6-TGDP-loaded RhoC protein was treated with redox agents inthe presence of free [³H]6-TGDP (10 μM). An association of RhoC proteinwith [³H]GDP to produce a RhoC-[³H]GDP complex will occur proportionallyafter RhoC releases 6-TGDP through the action of redox agents.Therefore, the fraction of the redox agent-mediated release of the bound6-TGDP per Rho protein over time can be deduced by determination of theRhoC-bound [³H]GDP, where the quantity of RhoC-bound 6-TGDP equals 1minus the determined fraction of the RhoC-bound [³H]GDP as described inSection (b). The estimated data associated with the quantity ofRhoC-bound 6-TGDP then was fitted to a one-phase exponential decay;Prism software was used to derive the rate of 6-TGDP dissociation fromRhoC.

(d) The effect of .NO₂ on the dissociation of 6-TGDP from RhoC wasdetermined by treating the RhoC-6-TGDP complex titration with variousconcentrations of [³H]GDP in the presence and absence of a reducingagent of either dithiothreitol (DTT) or β-mercaptoethanol. The data werefitted to a hyperbola with Prism Software to estimate the ^(app)K_(D) ofRhoC with 6-TGDP in the presence of [³H]GDP.

Effects of Thiopurines on SUM and RhoC-Overexpressed HME Cells in thePresence and Absence of a Redox Agent.

SUM149 cells and HME-RhoC have exhibited an invasive cell motility (FIG.14A). Unlike with SUM149 and HME-RhoC cells, SUM102 and HME cells thatare not overexpressed with RhoC do not exhibit cell motility. Theobserved RhoC-dependent cell motility of these SUM149 and HME-RhoC cellsis consistent with a previous study (van Golen et al. Mol. Cancer. Ther.1, 575-583 (2002)). Unlike the action of 6-TG on fast growing tumorcells (Heo et al. Biochemistry 49, 3965-3976 (2010)), the apoptoticeffect of 6-TG on SUM149 and HME-RhoC cells was minimally affected bytreatment with 6-TG for 3 days (FIG. 25A). The apoptotic effect of 6-TGon SUM149 and HME-RhoC cells also was minimal (FIG. 25B). This result issimilar to the effect of 6-TG on primary breast carcinoma in whichtreatment with 6-TG concentrations of less than ˜2 μM for 3 days hasminimal effect on the survival of primary breast carcinoma cells (Yamaneet al. Cancer Res. 67, 6286-6292 (2007)).

However, continuous treatment of SUM149 and HME-RhoC cells with 6-TG forthree or more consecutive days resulted in a gradual but significantdiminution of cell motility (FIG. 14A). Despite the diminution of thisactivity, the level of total RhoC expression in these SUM149 andHME-RhoC cells was not changed (FIG. 14A). Because cell motility iscoupled with the activity of RhoC but not the level of RhoA expression,this diminution of cell motility is likely correlated with the6-TG-mediated deregulation of RhoC activity.

Invasive cell motility also can be achieved by an overexpression of themutant RhoC C20S in HME (i.e., HME-C20S RhoC) cells (FIG. 14A). However,the effect of 6-TG on the motility of HME-C20S RhoC cells wasinsignificant compared with the effect of 6-TG on the motility of SUM149and HME-RhoC cells (FIG. 14A). The activity of, and total expression of,RhoC C20S also were unchanged by the treatment of HME-C20S RhoC with6-TG for three days (FIG. 14A). This result suggests the possibilitythat the RhoC residue Cys²⁰ somehow plays a role in the action of the6-TG-mediated diminution of the motility of SUM149 and HME-RhoC cells.

The cell viability and caspase activity of these SUM149, HMERhoC, andHME-C20S RhoC cells were minimally changed by treatment with the NOdonor alone (FIG. 25). A minor effect of the NO donor DETA/NO on theviability of head and neck squamous cell carcinoma (cell-line HNSCC) wasalso observed; however, this effect may depend on the type of cellinvolved (Azizzadeh et al. Laryngoscope 111, 1896-1900 (2001)). Theeffect of three days of continuous treatment with a NO donor—DETA/NO orGSNO—on the invasive cell motility and RhoC activity of these SUM149,HME-RhoC or HME-C20S RhoC cells was minimal (FIG. 14A). The motility ofboth SUM149 and HME-RhoC cells treated with a NO donor DETA/NO or GSNOinitially increased (10-30%) within 5 h, but after 10 h this enhancedmotility declined slightly below the original level. This initialactivation may be due to the redox response of RhoC because this proteinpossesses the GXXXXGK(S/T)C (SEQ ID NO:1) motif (Heo et al. J. Biol.Chem. 280, 31003-31010 (2005); Heo et al. Biochemistry 45, 14481-14489(2006)).

This notation is supported because the cellular activity of C20S RhoCwas unchanged after treatment of HME-C20S RhoC cells with a NO donor;the initial stimulatory effect of a NO donor on HME-C20S RhoC cells wasnot observed. Although the expression of RhoC was unchanged at thecellular level, essentially no RhoC activity was detected and completetermination of the invasive cell motility of SUM149 and HME-RhoC cellswas observed when these cells were treated for three days with both 6-TGand DETA/NO (or GSNO) (FIG. 14A). The viability and caspase activity ofSUM149 and HME-RhoC cells were minimally affected by treatment with 6-TGand DETA/NO (FIG. 25). This means that the 6-TG mediated termination ofthe invasive motility of these cells in the presence of the NO donorcannot be attributed to a change in the viability and/or induction ofapoptosis of these cells by 6-TG and DETA/NO. Just as with the singletreatment with 6-TG or a NO donor, the combined effect of 6-TG and a NOdonor is likely linked to the redox-sensitive GXXXXGK(S/T)C (SEQ IDNO:1) motif of RhoC. This reflects the comparative lack of a significanteffect of 6-TG with a NO donor on either the mutant C20S RhoC activityor the invasive cell motility of HME-C20S RhoC cells compared with theeffect of either 6-TG or a NO donor alone on the C20S RhoC or on thesesame HME-C20S RhoC cells, respectively (FIG. 14A).

Like with SUM149 cells, most of the invasive motility of SW480 andHCCLM3 tumor cells associated with RhoC (Wang et al. World J.Gastroenterol. 9, 1950-1953 (2003); Fiordalisi et al. Cancer Res. 66,3153-3161 (2006)) was terminated by treatment with 6-TG in combinationwith a NO donor for 3 days (FIG. 14B). Like with SUM149 cells (FIG.14A), the expression level of RhoC in these cells was unchanged, but theactivity of RhoC was drastically diminished when these cells weretreated with 6-TG and a NO donor (not shown). This result suggests that,regardless of tumor types, the RhoC-mediated invasive cell motility cancommonly be terminated by the action of 6-TG with a NO donor.

Analysis of RhoC 6-TGNP Adduct from Cells Treated with 6-TG and/or aRedox Agent.

To explore the underlying mechanism of the 6-TG-mediated inhibition ofthe motility of SUM149 and HME-RhoC cells in the presence of a redoxagent, RhoC protein was isolated from these cells treated with orwithout 6-TG in the presence or absence of a redox agent; this proteinthen was digested and peptides were analyzed with ESI-MS. A mass peak1481.6 Da., assigned to be a RhoC-derived peptide-6-TGDP disulfideadduct (TC²⁰LLIVFSK-6-TGDP) (SEQ ID NO:4) (FIG. 15B), was commonlyexhibited in co-immunoprecipitated (co-IPed) RhoC samples isolated fromboth

SUM149 cells (FIG. 15A, upper panel of the left column) and HME-RhoCcells treated with 6-TG (FIG. 15A, upper panel of the right column). Inthe presence of a NO donor, the peak assigned to be a RhoC-6-TGDP adductalso was increased, suggesting that a NO donor enhances the formation ofthe RhoC-6-TGDP adduct. However, this 1481.6 Da. mass peak was not foundin control cell samples that were untreated with 6-TG (FIG. 15A, lowerpanel of the left and right columns). This result suggests that 6-TGDP,which is derived from treated 6-TG, targets the Cys²⁰ side chain of theredox sensitive GXXXXGK(S/T)C (SEQ ID NO:1) motif of RhoC in SUM149 andHME-RhoC cells to produce a RhoC-6-TGDP adduct.

Distinctive to SUM149, RhoC mRNA expression in SUM102 cells has beenshown to be low (van Golen et al. Clin. Cancer. Res. 5, 2511-2519(1999)). However, the minimally expressed RhoC in SUM102 cells alsocould react with 6-TGDP to produce the 1481.6 Da. peak (FIG. 15A, middlepanel of the left column). Additionally, other Rho GTPases, such as RhoAand RhoB, possess the redox-sensitive GXXXXGK(S/T)C (SEQ ID NO:1) motifwith an identical sequence of TC²⁰LLIVFSK (SEQ ID NO:4). Hence, it alsois possible that the origin of the 1481.6 Da. peak from the SUM102sample can be derived from the reaction between 6-TGDP and theendogenously present RhoA and/or RhoB. Because C20S RhoC lacks theredox-sensitive Cys²⁰ residue, the RhoC-peptide adduct was not expectedto be detected in HME-C20S RhoC cells. However, a low intensity butdefinite 1481.6 Da. peak was also observed when HME-C20S RhoC cells weretreated with 6-TG (FIG. 15A, middle panel of the right column). As withSUM102 cells, the endogenously expressed RhoA and RhoB in HME-C20S RhoCcells may be a target of 6-TGDP and subsequently produce a RhoA- and/orRhoB-6-TGDP adduct.

Because cells maintain a ratio of GTP/GDP larger than 1 (Traut, T. W.Mol. Cell. Biochem. 140, 1-22) (1994)), the cellular concentration of6-TGTP also is likely to exceed that of 6-TGDP. However, ESI-MS analysishas detected a RhoC fragment TC²⁰LLIVFSK with 6-TGDP(TC²⁰LLIVFSK-6-TGDP) (SEQ ID NO:4) but not with 6-TGTP(TC²⁰LLIVFSK-6-TGTP) (SEQ ID NO:13) (FIG. 15A). This is likely becausethe γ phosphate of 6-TGTP is so unstable that during preparation of theESI-MSI sample, particularly the digestion at room temperature of theco-IPed RhoC fraction with trypsin, results in conversion of the 6-TGTPinto 6-TGDP that was covalently attached to the RhoC Cys²⁰ side chain.

Kinetic Properties of Rho GTPases with 6-TGNP in the Presence or Absenceof a Redox Agent.

To better understand the 6-TGNP-mediated inactivation mechanism of RhoCwith or without a redox agent, a redox-based biochemical analysis wasperformed for Rho GTPases, including RhoC and RhoA with 6-TGDP.

A competitive binding study shows that GDP bound to RhoA and RhoC can bedisplaced with 6-TGDP (FIG. 16A), similar to that of the Ras GDP with6-TGDP (Heo et al. Biochemistry 49, 3965-3976 (2010)). The RhoC andmutant RhoC bound GDP also can be competitively displaced with 6-TGDP(FIG. 16A). The binding affinities that 6-TGDP has for all of theseexamined Rho proteins are ˜2-fold weaker than that of GDP for Rhoproteins (FIG. 16A). However, the range of the determined values of thetrue dissociation constant (^(true)K_(D)) of Rho proteins for 6-TGDP,including C20S RhoC (FIG. 16A), nevertheless indicates that 6-TGDP has ahigh affinity for binding to Rho GTPases.

None of the redox agents tested was able to enhance GDP dissociationfrom C20S RhoC (FIG. 16B). Rates of the NO- or N₂O₃-mediated GDPdissociation from RhoC were minimal (FIG. 16B). However, .NO₂ enhancesdissociation of GDP from RhoC (FIG. 16B). These results were consistentwith a previous study suggesting that .NO₂, but neither NO nor N₂O₃,targets the GXXXXGK(S/T)C (SEQ ID NO:1) motif of the redox-sensitive RhoGTPases to enhance Rho GDP dissociation (Heo et al. J. Biol. Chem. 280,31003-31010 (2005)). .NO₂, but neither NO nor N₂O₃, enhancesdissociation of 6-TGDP from the redox inert C20S RhoC (FIG. 16C). Aprevious study (Heo et al. J. Biol. Chem. 280, 31003-31010 (2005))provides an explanation for this result in which .NO₂ targets the sulfuratom of the bound 6-TGDP, rather than the redox inert C20S RhoC protein,to produce the 6-TGDP-NO₂ adduct that can be degraded into5-guanidino-4-nitroimidazole diphosphate. Dissociation of 6-TGDP fromRhoC and mutant RhoC C20S by NO or N₂O₃ was minimal (FIG. 16C).

Because of the result associated with the action of .NO₂ on theGDP-bound RhoC combined with the 6-TGDP-bound C20S RhoC, it ispredictable that the target action of .NO₂ on a RhoC-6-TGDP complexwould be more effective; this is because both the ligand and receptor ofthe RhoC-6-TGDP complex, 6-TGDP and RhoC, are redox sensitive.Unexpectedly, however, a much slower rate of .NO₂-mediated 6-TGDPdissociation from RhoC was observed compared with that of 6-TGDPdissociation from C20S RhoC or GDP dissociation from RhoC (FIG. 16C). Tobetter understand this enigmatic result, the 6-TGDP-loaded RhoC waspretreated with .NO₂, and a competitive displacement of the preloaded6-TGDP with GDP was performed. Only a minimal fraction of the preloaded6-TGDP was dislodged with GDP (FIG. 16D). Dbs, the RhoC GEF, also wasunable to displace the bound 6-TGDP with GDP. This blockage ofdissociation of the bound 6-TGDP from RhoC was not observed when theRhoC-6-TGDP complex was untreated or pretreated with NO or N₂O₃. Themass peak 1481.6 Da. identified in cell samples treated with 6-TG (FIG.15A) also was found in this in vitro kinetic study sample of RhoC-6-TGDPthat was treated with .NO₂ and trypsin but not with either NO or N₂O₃.The 6-TGDP in the .NO₂-treated 6-TGDP C20S RhoC complex can be displacedwith GDP (FIG. 16D). The kinetic results with RhoC and mutant RhoC inconjunction with MS data suggests that the .NO₂-mediated formation ofthe RhoC Cys²⁰-6-TGDP disulfide adduct is linked to blockage of thedissociation of the 6-TGDP from RhoC in the presence and absence of Dbs.

A treatment of the .NO₂ pretreated-RhoC-6-TGDP complex with a reducingagent DTT enables the competitive displacement of RhoC-bound 6-TGDP withGDP (FIG. 16D). Another reducing agent, β-mercaptoethanol, also enhancesdisplacement of RhoC bound 6-TGDP with GDP. This is likely because DTTor β-mercaptoethanol reduces and thus disrupts the disulfide bondbetween 6-TGDP and the RhoC Cys²⁰ side chain. This unlinked 6-TGDP canthen be liberated from the RhoC protein, which is consistent withdisulfide blockage.

Potential Mechanism for the Formation of the RhoC-6-TGDP Adduct.

A hypothesis that the formation of the RhoC Cys²⁰-6-TGDP disulfideadduct results in the blockage of the dissociation of the bound 6-TGDPfrom RhoC is quite possible because the sulfur atom of the bound 6-TGDPis vicinal to the sulfur atom of the RhoC Cys²⁰ side chain (Dias et al.Biochemistry 46, 6547-6558 (2007)).

The results of an in vitro kinetic study also show that .NO₂ onlyenhances formation of the RhoC Cys²⁰-6-TGDP disulfide adduct. This isconsistent with previous studies showing that .NO₂ reacts with athiolate to produce a thiyl radical (Ford et al. Free Radic. Biol. Med.32, 1314-1323 (2002); Jourd'heuil et al. J. Biol. Chem. 278, 15720-15726(2003)). The thiyl radical formed also can react with a thiolate toproduce a disulfide radical anion (Xu et al. Chem. Rev. 107, 3514-3543(2007)). Because a disulfide radical anion is a strong reductant, itpredominantly reacts with O₂ to produce O₂.⁻ and a disulfide. On thebasis of these previous studies, a molecular mechanism is proposed forthe formation of the RhoC-6-TGDP disulfide adduct in the presence of.NO₂ (FIG. 17A). .NO₂ reacts with the RhoC Cys²⁰ side chain to produce aRhoC Cys²⁰ side chain thiyl radical. This RhoC radical then reacts withthe sulfur of the RhoC-bound 6-TGDP in a 6-thioxo form to give a RhoCCys²⁰ disulfide radical anion. This protein radical anion can bequenched by O₂ to produce a superoxide anion radical and the RhoC-6-TGDPdisulfide adduct. It is also possible that the thiyl radical formed canfurther react with another thiyl radical to produce a disulfidemolecule. Therefore, alternatively, if the 6-TGDP that possessesthiolate (a 6-sulfido form) is dominant, the sulfur atom of the bound6-TGDP will be a target of .NO₂ to produce a 6-TGDP thiyl radical. This6-TGDP radical then reacts with the RhoC Cys²⁰ side chain thiyl radicalto produce the RhoC-6-TGDP disulfide adduct. However, because the^(true)K_(D) value of RhoC for 6-TGDP deviated insignificantly from thevalue of RhoC for GDP, the state of the bound 6-TGDP is likely to be ina 6-thioxo form. Thus, formation of RhoC-6-TGDP disulfide via a directtargeting of the bound 6-TGDP is unlikely.

The nonradical-based process (FIG. 17B) also is possible. However,because formation of a disulfide bond was not detected between RhoC andthe bound 6-TGDP in the presence of a transition metal (i.e., Fe²⁺ orCu²⁺) but absence of .NO₂ under in vitro experimental conditions, thisnonradical-based mechanism is unlikely to occur.

All forms of 6-TGNP, such as 6-TGDP and 6-TGTP, have the same 6-TGmoiety; they differ from each other only in the number of phosphateslinked to the thio-nucleotide ribose. The proposed mechanism suggeststhat the results of kinetic analysis of 6-TGDP with RhoC shown in thisstudy are intrinsic to the chemical properties of the thiolate moiety of6-TG of 6-TGDP coupled with the RhoC Cys²⁰ side chain. Hence, thebiochemical features of other 6-TGNPs, such as 6-TGTP, with RhoC willnot differ significantly from those of 6-TGDP with RhoC (FIG. 16).

Example 4 Inhibitory Action of Thiopurine Drugs on RhoA GTPase and itsImplication in Smooth Muscle Cells Underlying Blood Vessels

Although misregulation of RhoA seems linked to major blood vesseldiseases (Rabinovitch, M. Toxicol. Pathol. 19, 458-469 (1991); Numaguchiet al. Circ. Res. 85, 5-11 (1999); Laufs et al. Circ. Res. 87, 526-528(2000); Alvarez de Sotomayor et al. Eur. J. Pharmacol. 415, 217-224(2001); Kuzuya et al. J. Cardiovasc. Pharmacol. 43, 808-814 (2004);Kontaridis et al. Circulation 117, 1423-1435 (2008)), no therapeuticagent exists that directly targets and inhibits RhoA activity (Oka etal. Br. J. Pharmacol. 155, 444-454 (2008)). 6-TGNP is shown to alsotarget and inhibit RhoA in A7r5 cells derived from rat aortic VascularSmooth Muscle Cells (VSMCs). This 6-TG-mediated inhibition of RhoA mayinduce vasorelaxation in blood vessels, because inactivation of RhoA wasshown to be coupled with the dephosphorylation of the myosin light chain(MLC) in A7r5 cells. Dephosphorylation of MLC is known to causevasorelaxation. The mechanism of action of TP drugs (as a form of6-TGNP) on RhoA inactivation likely is the same as that for TP drugs onRac1 inactivation; although 6-TGNP can target any known redox-sensitiveRho protein, such as Rac1 or RhoA, it specifically targets and inhibitsRac1 or RhoA via formation of a Rac1-6-TGNP adduct or RhoA-6-TGNPadduct, respectively, in T and A7r5 cells. Other findings with regard tothe action of TP drugs on Rac1 and RhoA GTPases include: (i) a novelanalog of 6-TP, 8-TP (i.e., 8-TG), can be converted into 8-thioguanosinephosphate (8-TGNP), which also targets and inhibits RhoA in A7r5 cellsvia formation of a RhoA-8-TGNP disulfide adduct; (ii) a redox agent,nitric oxide (NO), enhances the 6- or 8-TP-mediated blocking of RhoGTPase guanine nucleotide exchange (GNE). This enhancement may occur byfacilitating formation of the Rho protein-6- or 8-TGNP adduct; and (iii)finally, 6- or 8-TP aids dephosphorylation of the MLC and causesvasorelaxation in A7r5 cells.

Effects of 6-TPs on RhoA in A7r5 and T Cells.

The cellular expression levels of RhoA and of the MLC in A7r5 cells inthe presence or absence of 6-TG was not changed (FIG. 20A). However, asignificant portion of RhoA was inactivated within 3 days when A7r5cells were treated with 6-TG (FIG. 20B). A large portion of the MLC inA7r5 cells was observed to have been dephosphorylated after 3 daysfollowing treatment with 6-TG (FIG. 20B). Analyses using co-IP-basedESI-MS (FIG. 21) suggest that this result occurs because the thiolmoiety of 6-TGNP (cellularly converted from the treated 6-TG) reactswith the thiolate side chain Cys²⁰ of the RhoA GXXXXGK(S/T)C (SEQ IDNO:1) motif to produce a biologically inactive 6-TGNP-RhoA disulfideadduct. Taking these results into consideration, along with theRhoA/ROCK signaling associated with vasocontraction and dilation (FIG.23, Path A), and without being bound by theory, it is hypothesized that6-TPs target and inactivate RhoA. In turn, this results indephosphorylation of the MLC via a cascade of downregulation and thusupregulation of ROCK and the MLCP. Using co-IP-based ESI-MS, a6-TGNP-Rac1 disulfide adduct has been identified in activated T cellstreated with 6-TG (FIG. 21). This result in conjunction with previousstudies (Tiede et al. J. Clin. Invest. 111, 1133-1145 (2003); Poppe etal. J. Immunol. 176, 640-651 (2006)) suggests that the 6-TP mediatedinactivation of Rac1 in T cells is due to the formation of a 6-TGNP-Rac1disulfide adduct.

Examination of the Potential Target Specificity of 6-TPs on RhoA in A7r5Cells.

To examine whether the cellular targeting action of 6- or 8-TG on RhoAdepends on the action of Dbs and its target specificity, A7r5 cells weretreated with small interfering RNA directly against Dbs (Dbs siRNA) inthe presence and absence of 6- or 8-TG. The results show that thefraction of inactive RhoA and dephosphorylated form of the MLC in A7r5cells treated with both Dbs siRNA and 6- or 8-TG was smaller than thefraction of inactive RhoA and dephosphorylated form of the MLC inuntreated A7r5 cells. However, the fraction of inactive RhoA anddephosphorylated form of the MLC in A7r5 cells treated with both DbssiRNA and 6- or 8-TG was similar to that treated with only 6- or 8-TG(data not shown). The interpretation of these results is that theelimination of Dbs mRNA by siRNA results in loading failure of not only6- or 8-TGNP but also of regular GNP onto RhoA; the result is theinhibition of RhoA and dephosphorylation of the MLC regardless of thepresence of 6- or 8-TG. Hence, the current cell results using Dbs siRNAdo not constitute evidence of the role of Dbs in the action of 6- or8-TGNP on RhoA.

An in vitro kinetic study has been designed that can verify whether thetarget action of 6- or 8-TG on Rac1 hinges on the action of Vav and itstarget specificity. Although this proposed in vitro experiment is notcell based, it will aid in explaining the role of the GEF in the 6- or8-TG-mediated inactivation of Rac1 or RhoA.

Effects of a Redox Agent on the Formation of 6-TGNP-RhoA Adduct in A7r5Cells.

An endogenously released cellular redox agent (e.g., .NO₂ or O₂.⁻) couldbe involved in the formation of 6-TGNP-Rho protein adduct in cells. Thispossibility arises because a thiol (or thiolate) does not react withanother thiol except in the presence of a radical redox agent (e.g.,.NO₂ or O₂.⁻) (Heo et al. Biochemistry 45, 14481-44489 (2006)).Additional treatment of cells with an NO releasing agent (e.g.,diethylenetriamine/nitric oxide (DETA/NO); 10 μM for every 8 h), inaddition to 6-TG, synergistically increases the fraction of inactiveRhoA and the dephosphorylated form of the MLC (FIG. 20B). Because NOreleased from DETA/NO can be converted into .NO₂ in the presence of O₂,which enhances disulfide bond formation (Heo et al. Biochemistry 45,14481-14489 (2006)), the rate at which an NO donor enhances formation ofthe bound 6-TGNP-RhoA disulfide bond (FIG. 7) is hypothesized. MSanalysis also showed that the MS peak at 1476.6 Da, which represents the6-TGNP-RhoA adduct, increased when cells were treated with both 6-TG andNO. However, under experimental conditions, the intensity of the MS peakwas not truly quantitative. Thus, the intense MS peak for the6-TGNP-RhoA adduct does not necessarily indicate that NO enhancesformation of the 6-TGNP-RhoA adduct. Nonetheless, this enhancedformation of the 6-TGNP-RhoA adduct may further facilitatedephosphorylation of the MLC. However, because NO can down-regulate theMLCK via the sGC signaling pathway that populates the dephosphorylatedform of the MLC (FIG. 23, Path B), the synergistic action of a redoxagent may be partly because of the NO-mediated downregulation of theMLCK. This postulation of the MLCK downregulation is consistent with theresult that when cells were treated with only an NO donor (DETA/NO ≅10μM), a minimal fraction of RhoA was inactivated, but a sizable fractionof the MLC was dephosphorylated (FIG. 20B). Intriguingly, RhoA also canbe inactivated by an NO donor alone, but this inactivation requires arelatively higher concentration of the NO donor (i.e., DETA/NO >˜50 μM)(not shown). This redox agent concentration-dependent RhoA inactivationis likely because DETA/NO facilitates formation of a disulfide betweenRhoA Cys²⁰ and Cys16 that leads to inactivation of RhoA (Heo et al.Biochemistry 45, 14481-14489 (2006)).

Effects of 8-TG on RhoA.

A rapid accumulation of 6-TG in the DNA of fast-growing cells (i.e.,tumors) triggers the action of MMR, inducing apoptosis (Heo et al.Biochemistry 49, 3965-3976 (2010)). However, because of the gradual rateof accumulation of DNA 6-TG in slow-growing cells, such as A7r5 cells,the observed cell apoptosis is relatively minimal compared with whatoccurs in fast-growing tumor cells. Nevertheless, this minimal6-TG-mediated apoptosis may still cause a certain degree of cytotoxicity(i.e., an increase in cell viability up to ˜5%). To develop a betteragent to minimize potential cytotoxicity, the effects of various 6-TPisomers on A7r5 cells have been screened, such as 8-TG(2-amino-8-mercapto-1H-purin-6(9H)-one, see FIG. 4) and 7-thioguanine(2-amino-7-mercapto-8,9-dihydro-1H-purin-6(H)-one). Of the severalisomers screened, 8-TG most effectively inhibits RhoA activity anddephosphorylates the MLC (not shown). As with 6-TGNP (FIG. 21),co-IP-based ESIMS analysis indicates that the thiol moiety of 8-TGNP(cellularly converted from the treated 8-TG) reacts with the thiolateside chain Cys²⁰ of the RhoA GXXXXGK(S/T)C (SEQ ID NO:1) motif toproduce a biologically inactive 8-TGNPRhoA disulfide adduct (FIG. 22).

The structural feature of the nucleotide-binding pocket of RhoA explainsthe effective inhibitive action of 8-TG on cells when the distancebetween the sulfur atom of the RhoA Cys²⁰ side chain and the sulfur atomat the C8 position of base (˜3.6 Å) is shorter than that of the sulfuratom of the RhoA Cys²⁰ side chain and the sulfur atom at the C6 positionof base (˜6.9 Å) (FIG. 12) Ohara et al. J. Biol. Chem. 273, 9656-9666(1998)). Hence, the superior action of 8-TGNP is likely becausestructural cellular conditions may favor formation of a disulfide bondbetween the RhoA Cys²⁰ side chain and 8-TGNP over a similar bond with6-TGNP.

Intriguingly, although 6-TG can induce minimal cytotoxicity in A7r5cells, 8-TG did not alter the viability of A7r5 cells under experimentalconditions (FIG. 24). While the aim is not to characterize thecellular/mechanical reason for the minimal cytotoxicity of 8-TG comparedto that of 6-TG, the No. 6 position of the sulfur atom of 6-TG, but notthe No. 8 position of the sulfur atom of 6-TG, in DNA likely triggersMMR to induce apoptosis. Evading the DNA MMR can be of benefit for apotential drug (e.g., 8-TG) that targets a protein such as RhoA. Thiswould be because, compared with 6-TPs, it consequently could not havethe cytotoxicity associated with MMR-induced apoptosis. However, evadingthe DNA MMR could be a disadvantage for a drug aimed at inducingapoptosis. In fact, a therapeutic effect of 6-TPs is rooted instimulation of the DNA MMR (Lage et al. J. Cancer Res. Clin. Oncol. 125,156-165 (1999); Yan et al. Clin. Cancer Res. 9, 2327-2334 (2003); Karranet al. Nat. Rev. Cancer 8, 24-36 (2008)). More 6-TP can be incorporatedinto the DNA of fast-growing tumors (e.g., acute lymphoblastic leukemia)than into the DNA of normal cells. Hence, more DNA MMR-induced apoptosiscan occur in tumor cells (e.g., acute lymphoblastic leukemia) than innormal cells. Nonetheless, 8-TG and its isomers may have been ignored aspossible therapeutic agents (i.e., as antitumor agents) because 8-TGdoes not effectively induce MMR-mediated apoptosis.

Example 5 Immunosuppressive Effects of Thiopurine Drugs

The primary objective of this example is to examine the chemistry-basedmolecular mechanism by which commercially available thiopurine (TP)drugs may block T-cell activation via inhibition of Rac1, therebysuppressing the immune response. The results of these studies form abasis for the development of TP-based chemotherapeutic agent(s) forautoimmune disorders.

The cellular and biochemical effect of 6-TG in combination with orwithout a redox agent on Rac1 was examined. The cellular expressionlevel of Rac1 and ERM in T cells in the presence or absence of 6-TG wasnot changed (FIG. 26A). However, a significant portion of Rac1 wasinactivated within 3 days when T cells were treated with 6-TG (FIG.26B). A large portion of ERM in T cells was observed to have beenphosphorylated after three days following treatment with 6-TG (FIG.26B). Analyses using co-immunoprecipitation-based electrosprayionization mass spectrometry (co-IP-based ESI-MS) (See FIG. 21) suggestthat this result occurs because the thiol moiety of 6-TGNP (cellularlyconverted from the treated 6-TG) reacts with the thiolate side chainCys²⁰ of the Rac1 GXXXXGK(S/T)C (SEQ ID NO:1) motif. This reactionproduces a biologically inactive 6-TGNP-Rac 1 disulfide adduct. Takinginto consideration these results along with previous study results(Tiede et al. J. Clin. Invest. 111, 1133-1145 (2003); Poppe et al. J.Immunol. 176, 640-651 (2006)), without being bound by theory, it isbelieved that 6-TPs target and inactivate Rac1 via formation of the6-TGNP-Rac1 disulfide adduct (FIG. 29, Path B). In turn, this results inblockage of the dephosphorylation of ERM (FIG. 29, Path D) and thusinactivates T cells.

Effects of a Redox Agent on the Formation of 6-TGNP-Rac1 Adduct inCells.

An endogenously released cellular redox agent could be involved in theformation of 6-TGNP-Rac1 adduct in cells (FIG. 29, Path B). Thispossibility arises because a thiol (or thiolate) does not react withanother thiol unless a radical redox agent (e.g., .NO₂ or O₂{tilde over(.)}⁻) is present (Heo et al. Biochemistry 45, 14481-14489 (2006)).

Additional treatment of cells with an NO releasing agent (e.g.,diethylenetriamine/nitric oxide (DETA/NO); 10 μM for every 8 h), inaddition to 6-TG, synergistically increases the fraction of inactiveRac1 and also increases the blockage of the dephosphorylated form of ERM(FIG. 29B). Because NO released from DETA/NO can be converted into .NO₂in the presence of O₂, which enhances formation of the disulfide bond(Heo et al. Biochemistry 45, 14481-14489 (2006)), it is believed therate at which an NO donor enhances formation of the bound 6-TGNP-Rac1disulfide bond (see FIG. 7). MS analysis also showed that the MS peak at1476.6 Da, which represents the 6-TGNP-Rac1 adduct, increased when cellswere treated with both 6-TG and NO. However, under experimentalconditions, the intensity of the MS peak was not truly quantitative;consequently, the intensity of the MS peak for the 6-TGNP-Rac1 adductdoes not necessarily indicate that NO enhances formation of the6-TGNP-Rac1 adduct. Nonetheless, this enhanced formation of the6-TGNP-Rac1 adduct may further facilitate blockage of thedephosphorylation of ERM.

Effects of 8-TG on Rac1:

A rapid accumulation of 6-TG in the DNA of fast growing cells (i.e.,tumors) triggers the action of MMR, inducing apoptosis (Heo et al.Biochemistry 49, 3965-3976 (2010)). However, because of the gradual rateof accumulation of DNA 6-TG in slow growing cells, such as T cells andprimary epithelial cells, the observed cell apoptosis is relativelyminor compared with what occurs in fast growing tumor cells. However,this minimal 6-TG-mediated apoptosis may still cause a degree ofcytotoxicity. To develop a better agent to minimize this potentialcytotoxicity, the effects of various 6-TP isomers on T cells have beenscreened, such as 8-TG (2-amino-8-mercapto-1H-purin-6(9H)-one) and7-thioguanine (2-amino-7-mercapto-8,9-dihydro-1H-purin-6(H)-one). Of theseveral isomers screened, 8-TG most effectively inhibits Rac1 activityand blocks the dephosphorylation of ERM (not shown). As with 6-TGNP(FIG. 21), co-IP-based ESI-MS analysis indicates that the thiol moietyof 8-TGNP (cellularly converted from the treated 8-TG) reacts with thethiolate side chain Cys¹⁸ of the Rac1 GXXXXGK(S/T)C (SEQ ID NO:1) motifto produce a biologically inactive 8-TGNP-Rac1 disulfide adduct (FIG.27).

The structural feature of the nucleotide-binding pocket of Rac1 (PDB 1MH1) explains the effective inhibitory action of 8-TG on cells when thedistance between the sulfur atom of the Rac1 Cys¹⁸ side chain and thesulfur atom at the C8 position of base (˜3.6 Å) is less than thedistance of the sulfur atom of the Rac1 Cys¹⁸ side chain from the sulfuratom at the C6 position of base (˜6.9 Å) (FIG. 28) (Hirshberg et al.Nat. Struct. Biol. 4, 147-152 (1997)). Hence, the closer proximity ofthe Rac1 Cys¹⁸ side chain and 8-TGNP is why they are the more likelypair to form a disulfide bond.

These studies show that 6-TP drugs (as a form of 6-TGNP) target andinhibit Rac1 via formation of a Rac1-6-TGNP adduct in T cells. Thesestudies also show that 8-TP (i.e., 8-TG), an analog of 6-TP drugs, canbe converted into 8-thioguanosine phosphate (8-TGNP) that targets andinhibits Rac1 in T cells. Without being bound by theory, it is believedthat the formed disulfide adduct blocks the Rho guanine nucleotideexchange factor (GEF)-mediated guanine nucleotide exchange (GNE) of Rhoprotein, resulting in accumulation of an inactive Rho protein.Intriguingly, a redox agent, nitric oxide (NO), enhances the TP-mediatedblocking of Rho GTPase GNE. This enhancement may occur via facilitationof the formation of the Rho protein-6 or 8-TGNP adduct. Without beingbound by theory, a model mechanism of 6-TP-mediated immunosuppression isshown in FIG. 29. This proposed mechanism explains the previousobservation in which the observed blockage of the Vav-mediated GNE ofthe 6-TGNP-bound Rac1 (Tiede et al. J. Clin. Invest. 111, 1133-1145(2003); Poppe et al. J. Immunol. 176, 640-651 (2006)) (FIG. 29, path C)is because of the reactivity of 6-TGNP with the Rac1 Cys¹⁸ to produce a6-TGNP-Rac1 adduct that in turn blocks the action of Vav for Rac1 GNE.

While there have been shown and described what are presently believed tobe the preferred embodiments of the present invention, those skilled inthe art will realize that other and further embodiments can be madewithout departing from the spirit and scope of the invention describedin this application, and this application includes all suchmodifications that are within the intended scope of the claims set forthherein. All patents and publications mentioned and/or cited herein areincorporated by reference to the same extent as if each individualpatent and publication was specifically and individually indicated ashaving been incorporated by reference in its entirety.

I claim:
 1. A pharmaceutical composition for inhibiting aredox-sensitive GTPase protein comprising an effective amount of aredox-sensitive purine compound, an effective amount of a redox agentand an acceptable carrier, wherein the redox-sensitive purine compoundis selected from the group consisting of a 6-thiopurine compound and an8-thiopurine compound, wherein the GTPase protein comprises aGXXXXGK(S/T)C motif and the GTPase protein is selected from the groupconsisting of a Rho family GTPase and a Rab family GTPase.
 2. Thecomposition of claim 1, wherein the 6-thiopurine compound is6-thioguanine and the 8-thiopurine compound is 8-thioguanine.
 3. Thecomposition of claim 1, wherein the redox-sensitive purine compound isselected from the group consisting of 8-thioguanine(2-amino-8-mercapto-1H-purin-6(9H)-one), 8-methylthioguanine(2-amino-8-(mercaptomethyl)-1H-purin-6(9H)-one), 7-methylthioguanine(2-amino-7-(mercaptomethyl)-8,9-dihydro-1H-purin-6(H)-one),7-thioguanine (2-amino-7-mercapto-8,9-dihydro-1H-purin-6(H)-one),azathioprine (AZA), 6-mercaptopurine (6-MP); and 6-thioguanine (6-TG).4. The composition of claim 1, wherein the redox-sensitive purinecompound is selected from the group of compounds of formula I, wherein:

R₁ is selected from the group consisting of C6-thioxo (C6=S; incombination with C6 of the purine base); C6-thiol (C6-SH; in combinationwith C6 of the purine base); C6-selenal (C6=Se; in combination with C6of the purine base); C6-selenol (C6-SeH; in combination with C6 of thepurine base); C1-4 straight chain or branched chain alkyl, alkenyl, oralkynyl, wherein C1-4 are unsubstituted, singly substituted or multiplysubstituted, wherein the substituents are selected from the groupconsisting of thiol, thioxo, selenal, selenol, hydroxyl, halogen, amino,ketone, alkoxy, aldehyde and carboxylic acid; OH; and O; wherein R₂ isselected from the group consisting of H; thiol (SH); selenol (SeH); C1-4straight chain or branched chain alkyl, alkenyl, or alkynyl, whereinC1-4 are unsubstituted, singly substituted or multiply substituted,wherein the substituents are selected from the group consisting ofthiol, thioxo, selenal, selenol, hydroxyl, halogen, amino, ketone,alkoxy, aldehyde and carboxylic acid; NH₂; and OH; wherein R₃ isselected from the group consisting of H, NH₂ and OH; with the provisothat at least one of R₁ or R₂ is a moiety that comprises aredox-sensitive functional group selected from the group consisting ofthioxo, thiol, selenal and selenol.
 5. The composition of claim 1,wherein the redox agent is selected from the group consisting of nitricoxide, nitrogen dioxide, dinitrogen trioxide, superoxide anion radical,hydrogen peroxide, carbonate radical, and an agent which stimulates theproduction of a redox agent.